Influence of lipid composition on opsonophagocytosis of liposomes

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of lipid composition on opsonophagocytosis of liposomes H.M. Pate1

Department

of Biochemistry,

Charing Cross and Westminster Medical London W6 8RF

School, Fulham Palace Road,

Introduction

Clearance by the RES

There are two completely opposing reasons for wanting to inject liposomes intravenously into man and animal. The first is to target the carriers selectively to a specific organ of the reticuloendothelial system (RES) and the second is to avoid the RES and direct the carriers to a site, or sites, other than the RES. Both of these aims can be achieved by a proper understanding of the processes of liposome recognition and subsequent uptake by mononuclear phagocytes in vivo, so that liposomes of the desired affinity for macrophages can be prepared. There are two possible ways in which liposomes may interact with macrophages. One depends on direct interaction of liposomes with cell surface and the other involves their interaction with serum proteins. These proteins can be opsonins or dysopsonins which may promote or suppress phagocytosis of liposomes, respectively. On exposure to blood, liposomes interact with plasma proteins (Bonte and Juliano, 1986; Bonte et al., 1987), and this process is thought to determine their recognition by mononuclear phagocytes. Purified serum opsonins such as immunoglobulins, complement components, fibronectin, C-reactive protein, tuftsin, have been used to opsonize liposomes under the experimental conditions and promote their phagocytosis in vitro as well as in vivo (reviewed in Patel, 1991). However, it is not clear to what extent these opsonins interact with liposomes in the circulation and what role they play in promoting phagocytosis of the circulating liposomes. The diversity and complexity of the process of opsonization of liposomes in the circulation make it difficult to assess the role of various proteins in the uptake of vesicles by the RES. A large number of factors govern the ability of liposomes to interact with serum proteins, and among these factors, physicochemical properties of both proteins and liposomal surface play an important role. The properties such as hydrophilicity, fluidity, charge and size of liposomes are easily manipulated by selecting appropriate phospholipids and lipid composition of liposomes.

The clearance of liposomes depends on the rate of degradation in the circulation and uptake by the RES. It has been suggested (Gregoriadis et al., 1983 ; Gregoriadis, 1988) that the vesicles having greater stability in blood have a longer half-life in the circulation. It is interesting to note that factors which make liposomes stable in blood also reduce their uptake by the RES. The destabilizing effect of serum components, in particular high-density lipoproteins (Scherphof et al., 1978; Allen, 1981) is minimized by making liposomal bilayers more rigid by inclusion of cholesterol and long-chain saturated phospholipids. The increased cholesterol content of liposomes also reduces the uptake of the vesicle by the RES particularly in the liver (Pate1 et a1.j i983, Gregoriadis, 1988). The presence of cholesterol increases the packing and rigidity of the bilayer when lipid would normally be above its critical temperature. Protein/vesicle interaction depends on the fluidity and packing of liposome bilayers, and the increased packing and rigidity of the bilayers retard penetration of proteins into the liposome membrane. Similarly solid liposomes, such as those composed of distearoyl phosphatidylcholine or sphingomyelin, are resistant to the destabilizing effects of serum components and have a reduced uptake into the liver (Ellens et al., 1981, Senior and Gregoriadis, 1982). These effects of different lipid composition of liposomes on blood clearance and uptake by the RES are believed to be due to the different degree of opaonization of the vesicles in the circulation. The electrophoretlc studies of cholesterol-poor (i.e. 20 mol % cholesterol content) and cholesterolrich (50 mol % cholesterol) liposomes opsonized with serum demonstrate that more proteins are associated with cholesterol-poor than with rigid cholesterolrich liposomes (Moghimi and Patel, 1988). When these liposomes are injected intravenously, cholesterol-poor liposomes are localized much more in the liver than cholesterol-rich, which are localized much more in spleen than cholesterol-poor liposomes

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(Pate1 et al., 1983; Pate1 and Moghimi, 1990). In vitro studies (Moghimi and Patel, 1988) have shown that Kupffer cells avidly take up cholesterol-poor but not cholesterol-rich liposomes in the presence of serum, whereas splenic phagocytic cells take up preferentially cholesterol-rich rather than cholesterolpoor liposomes. These results suggest that serum contains opsonins specific for hepatic and splenic phagocytic cells having different affinities for cholesterol-rich and cholesterol-poor liposomes. So far, very little is known about the properties of these opsonins (Moghimi and Patel, 1989; Pate1 and Moghimi, 1990). Liver-(Kupffer cell)-specific opsonin is a heat-stable proteinaceous macromolecule, and heating or freezing and thawing of serum cause enhancement in its activity, whereas spleen-specific opsonin is a heat-labile proteinaceous macromolecule which loses part of its activity on freezing and thawing. The activities of both opsonins are regulated by dialysable factors present in serum. These factors have opposite effects on the two opsonins. Their removal from the serum causes enhancement of liverspecific opsonic activity, whereas it suppresses spleenspecific activity. The dialysable factor that regulates liver-specific opsonic activity in serum is now identified as calcium, whereas calcium has no effect on the spleen-specific activity (Moghimi and Patel, 1990). The properties of bone marrow-specific opsonin are very similar to those of spleen-specific opsonin, and the properties of lung- and liver-specific opsonins are very similar (Pate1 and Moghimi, 1990). The tissue-specific opsonic activities are also found in serum of rabbit, calf and man and are not speciesspecific. From our attempts to purify these opsonins, we found that liver-specific opsonin is not immunoglobulin, component of complement, fibronectin or C-reactive protein. However, its identity is not known. We find that partially purified fraction loses its activity fast, whereas spleen-specific opsonin activity seems to be contributed by two separate factors and one of them is complement. Cholesterol-rich liposomes are indeed potent complement activators (Alving and Richards, 1983 ; Chonn et al., 1991) and promote complement protein/liposome interaction (Chonn et al., 1991). Thus, liposomes of differing surface characteristics attract different arrays of plasma proteins, and their interaction with liposomes may vary in quantity and conformation. And it is this difference which may account for the different patterns in their blood clearance and tissue distribution. Furthermore, phagocytic cells from different RE organs seem to take up liposomes by different mechanisms (reviewed in Patel, 1991). Neither liver- nor spleen-specific opsonins have affinity for liposomes with longer halflife in the circulation prepared from solid-phase phospholipids, and it has been suggested that these lipo-

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somes interact with serum proteins which have a dysopsonic effect and thus retard their uptake by the RES (Moghimi and Patel, 1989a). Opsonization of negatively charged liposomes

Among various physicochemical properties, the surface charge of liposomes is an important factors which overrides the state of membrane stability in determining the rate of uptake of vesicles by the RES. Negatively charged liposomes are removed more rapidly from the circulation and localized much more in liver, spleen and bone marrow, and are effectively trapped in the lung than neutral or positively charged liposomes (Fidler and Schroit, 1986). However, all negatively charged phospholipids do not have similar effects on liposomes clearance by the RES. Liposomes containing negatively charged phosphatidylserine (PS), phosphatidylglycerol (PG) and glycolipids and other negatively charged phospholipids are rapidly taken up by the RE organs, whereas negatively charged ganglioside G,,-, sulphatide- or phosphatidylinositol-containing vesicles are retained in the circulation for a much longer time and are poorly taken up by the RES (Gabizon and Papahadjopoulos 1988 ; Allen et al., 1989). The mechanism behind these diverse effects of negatively charged phospholipids is not clear. The opsonization of negatively charged liposomes will be influenced by the polar headgroups and location of negative charge of the molecule within the lipid bilayers. The negative charge group on PS, PC, phosphatidic acid and dicetyl phosphate are, for instance, exposed and thus allow direct interaction between the negatively charged group on liposome surface and plasma proteins or cell surface, whereas the negative charge in ganglioside (G,,) and PI is shielded by a bulky hydrophilic carbohydrate group present in these molecules. The negative charge for example, in the case of G,,, is shielded from the surface by the presence of two neutral sugars, while this is not the case with other gangliosides, Gota, Gr,,, and G,, which produce liposomes with a shorter halflife in comparison to G,, -containing liposomes (Allen et al., 1989). It has been suggested that the shielding of the negatively charged group prevents opsonization of G,, liposomes and hence gives longer half-life in the circulation (Gabizon and Papahadjopoulos, 1988; Allen et al., 1989). Liposomes with shielded negatively charged groupas may act as neutral liposomes as fars their interaction with serum proteins is concerned. But neutral liposome surface acquires the negative charge on exposure to blood by interacting with serum proteins, particularly crZmacroglobulin which is known to promote phagocytosis of particles (Black and Gregoriadis, 1976; Molnar et al., 1977). Thus, the shielding of a negatively charged group alone cannot prevent the

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opsonization of liposomes, but it seems that it is a synergistic effect of the rigid bilayer and carbohydrate residues which prevents their opsonization. The presence of carbohydrate molecules makes the liposome surface hydrophilic with which serum proteins interact poorly, as in the case of PEG-coated liposomes (reviewed in Patel, 1991). However, the possibility that these liposomes may interact with certain serum proteins which act as dysopsonins and suppress their uptake by the RES should not be discarded. Liposomes composed of net negatively charged phospholipids, such as PS or PG, activate the complement system (Alving and Richards, 1983 ; Chonn et al., 1991) and, depending on the lipid composition of the vesicles, C3b and iC3b complement components are deposited on liposomes (Loughrey et al., 1990). C3b may play an important role in the initial rapid phase of in vivo clearance of negatively charged liposomes, and the rate of clearance of liposomes will depend on their ability to activate the complement system. However, our results indicate that complement does not play any role in uptake of liposomes in liver (Moghimi and Patel, 1989; Pate1 and Moghimi, 1990), and furthermore, complement by itself plays only a limited role in phagocytosis of the synthetic particles (Saba, 1970). Hence, the search for the identification of other factors which may interact with liposomes in the circulation and promote their phagocytosis in various RES organs should go on.

References Allen, T.M. (1981),A study of phospholipidinteraction betweenhigh-densitylipoproteinsand smallunilamellar vesicles.Biochem. biophys. Acfu. (Amst.), 640, 385-397. Allen, T.M., Hansen,C. & Rutledge,J., (1989),Liposomes with prolongedcirculationtimes: factorsaffecting uptake by reticuloendothelialand other tissue.Biochim. biophys. Acta (Amst.), 981, 27-35. Alving, C.R. & Richards, R.L. (1983), Immunologic aspectsof liposomes,in “Liposomes” (Ostro, M.) (pp. 209-287).Marcel Dekker, New York. Black, C. & Gregoriadis,G., (1976), Interaction of liposomeswith blood plasmaproteins. Biochem. Sot. Trans., 4, 253.

Bonte, F. & Juliano, R.L., (1986), Interaction of liposomes with serumproteins.Chem.Phys. Lipids, 4Q,373-393. Bonte, F., Hsu, M.J., Papp, A., Wu, K., Regan,S.L. & Juliano, R.L. (1987), Interaction of polymerizable phosphatidylcholinevesicleswith blood components: relevance to biocompatibility. Biochim. biophys. Acta., 900, l-9. Chonn, A., CuBis,P.R. & Devine, D.V., (1991),The role of surfacechargein the activation of the classicaland

alternative pathways of complement by liposomes. J. Immunol., 146, 42344241.

Ellens, H., Morselt, H. & Scherphof, G. (1981). In vivo

fate of large unilamellar sphingomyelin-cholesterol

liposomes after intraperitoneal and intravenous injec-

tion into rats. Biochim. biophys. Acta (Amst.), 674, 10-18. Fidler, I.J. & Schroit, A.J. (1986),Macrophage recognition of self from nonself: implicationsfor the interaction of macrophageswith neoplasticcells. Symp. Fund. Cant. Res., 38, 183-207. Gabizon, A. & Papahadjopoulos, D. (1988),Liposomeformulationswith prolongedcirculation time in blood and enhanceduptake by tumours. Proc. nut. Acad. Sci., (Wash.), 85, 6949-6953. Gregoriadis, G., (1988), Fate of injected liposomes,in “Liposomes as drug carriers, Recent Trends & Progress”(GregoriadisG.,) pp. 3-19.John Wiley & Sons, Chichester. Gregoriadis,G., Kirby, C. & Senior, J. (1983),Optimization of liposomebehavior in vivo. Biol. Cell., 47, 11-18. Loughrey, H.C., Bally, M.B., Reinish, L.W. & Cullis, P.R., (1990), The binding of phosphatidylglycerol liposomesto rat plateletsis mediatedby complement. Thromb. Haemostasis.64, 172-179. Moghimi, S.M. & Patel, H.M. (1988), Tissue-specificopsoninsfor phagocytic cellsand their different affinity for cholesterol-richliposomes.FEBS Letters, 233, 143-147. Moghimi, S.M. & Patel, H.M. (1989),Differential properties of organ-specificserumopsoninsfor liver and spleenmacrophages. Biochim. biophys.Acta (Amst.), 984, 379-383. Moghimi, S.M. & Patel, H.M. (1989a),Serumopsonins and phagocytosisof saturatedand unsaturatedphospholipid liposomes.Biochim. biophys.Acta (Amst.), 984, 384-387. Moghimi, S.M. & Patel, H.M. (1990),Calciumasa possible modulatorof Kupffer cell phagocytic function by regulating liver-specific opsonic activity. Biochim. biophys. Actu (Amst.), 1028, 304-311. Molnar, J., McLain, S., Allen, C., Gara, A. & Gelder, F., (1977),The role of a a2-macroglobulinof rat serum in the phagocytosisof colloidal particles. Biochim. biophys. Acta (Amst.), 493, 37-45. Pate], H.M. (1991),Serumopsoninsand liposomes:their interaction and opsonophagocytosis.CRC Critical Reviewsin Therap. Drug Carrier System(in press). Patel, H.M. & Moghimi, S.M. (1990),Tissue-specificopsoninsand interaction with liposomes,in “Targeting of drugs: optimisationstrategies”. (Gregoriadis,G.) NATO Advanced Study Institute Serie, (pp. 87-94).

Plenum Press, New York. Patel, H.M., Tuzel, N.S. & Ryman, B.E. (1983), Inhibitory effect of cholesterolon the uptake of liposomes by liver and spleen.Biochim. biophys.Acta (Amst.), 761, 142-151. Saba, T.M., (1970), Physiology and physiopathology of the reticuloendothelialsystem.Arch. Intern. Med., 126, 1031-182. Scherphof, G., Roerdink, G., Waite, M. & Parks, J. (1978), Disintegration of phosphatidylcholineliposomesin plasmaas a result of interaction with highdensitylipoproteins.Biochim. biophys.Actu (Amst.), 542, 296-307. Senior, J. & Gregoriadis, G. (1982), Stability of small unilamellar liposomesin serumand clearancefrom the circulation: the effect of the phospholipidsand cholesterol components. Life Sciences,30,2123-2136.

245 r)ISCUSSION

G. Cregoriadis : The uptake of liposomes and entrapped proteins by hepatic and splenic macrophages in vivo (as well as by peritoneal macrophages in vitro) by the lysosomal pathway was established in the early 70’s. In retrospect, it is not surprising that soon afterwards, in 1974, Iiposomes were found (albeit surreptitiously~ to act as immunologica adjuvants (Gregoriadis, 1990). Much work has been carried out in the intervening years to extend the immunoadjuvant activity (humoral and cell-mediated immunity) of liposomes, given by a variety of routes to a wide range of bacterial, viral, protozoan, tumoral and other antigens. Further protection was observed in experimental models of animals immunized against disease. Yet, adjuvanticity was shown to vary profoundly not only among antigens tested but also for the same antigen in different hands. Clearly, in terms of the future of liposomes in vaccine formulations, this is unacceptable. Attempts have therefore been made to optimize liposomal adjuvanticity by controlling a number of liposomal structural characteristics and/or incorporating into liposomes a variety of immunomodulators (e.g. lipopolysaccharide, MDP derivatives and cytokines). Although some guid, lines of what is best for liposomal vaccines are now slowly emerging (Gregoriadis, 1990), the ideal formulation (if this is eventually to be) is far from being established. This state of affairs is not unique to liposomes. Other promising new-generation adjuvants such as the ISCOM and block copolymers have similar problems. The obvious way to optimize liposomal immunoadjuvant activity is one based on understanding how the system works in vivo. In view of the complexity and variability of both liposomes and their interaction with the biological milieu, acquisition of relevant knowledge is expected to be tortuous. Nevertheless, a first step has been made, namely the demonstration by van Rooijen’s and other groups that interaction of liposomes with macrophages is a prerequisite for adjuvanticity to occur in vitro. It is quite proper, therefore, that the importance of macrophages in this respect forms part of the present Forum, Study of liposomal adjuvanticity in vitro is a less complicated path to follow, and recently, considerable related progress has been made. Much of this progress is aired in the Forum. It includes discussions ranging in topics from the role that a variety of APC (antigen-presenting cells) play in the processing of liposome-entrapped antigens, the significance of antigen localization on the liposomal surface and the possible co-operation of APC in terms of antigen

presentation, to achieving class-I-MHC-mediated presentation of liposomal-entrapped antigens. No doubt, such experiments will prove useful in elucidating and perhaps controlling immune responses to liposomal antigens. I would like to briefly comment here on potential difficulties with translating in vitro observations into ways of tailoring liposomal adjuvanticity in vivo. Firstly, it is to be expected that liposomes incorporating antigen would, on injection, be coated with one or more proteins present in biological fluids. Would such coating influence the interaction of liposomes with APC? Would, for instance, proteincoated fusogenic liposomes perform their task of delivering antigens into the cytoplasm of APC? Would some of these adsorbed proteins facilitate interaction of the vesicles with APC which normally ignore the vesicles in vitro? If an antigen is located on the bilayer’s surface, would its presence diminish or even prevent the adsorption of proteins onto the bilayer ? Secondly, some of the injected liposomes, depending on their lipid composition and other characteristics, are expected to disintegrate gradually releasing antigen extracellularly. At the same time there would be vesicle uptake locally by infiltrating APC and also by APC in the lymphoid organs. In short (and in contrast to the in vitro situation), what was a homogenous, well-characterized vesicle-antigen population in the test tube is certain to end up in vivo as a kaleidoscope of entities. These would include free antigen, partly disrupted vesicles still containing some of the antigen and intact vesicles, with all entities migrating to various parts of the body, e.g. the lymphatic system, blood circulation and the liver, spleen and bone marrow. In view of such events, one can again ask several questions. At what stage(s) of such continually changing fate of the original liposomes does adjuvanticity occur, and is it influenced by more than one of the forms of antigen in existence? What is the influence on liposomal antigen fate of parameters as diverse as vesicle size, amounts of lipid injected, bilayer composition, surface charge, lamellarity and antigen localization? Thirdly, liposomes are expected to enter (mostly) macrophages by endocytosis to end up in the lysosomes. However, how certain are we that a small proportion does not also interact with dendritic or interdigitating cells? If so, is it not possible that such interaction, minor as it may be, plays a decisive role in adjuvanticity? And how about the events occurring from the moment liposomes enter cells to the time of their eventual disintegration in the lysosomes? Are we to assume that all liposomes are trans-

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ported intact and in isolation to their final intracellular destination? Is it not possible that some, depending on, say, composition or the physiological state of the cell, escape transporter vacuoles to reasch the cytoplasm? Events can hardly be idealized and should be expected to deviate, at least to some extent. The final and perhaps most urgent question is about priorities. On the one hand, there is unequivocal evidence of liposomal adjuvanticity and a plethora of ways to improve it so as to inspire confidence in those interested in using the system in vaccine formulations. Advances in liposome technology to ensure vesicle stability, reproducibility and largescale production have helped considerably to strengthen such confidence. On the other hand, we may be potentially faced with the herculean task of elucidating mechanisms as outlined already before the use of the system in vaccines can be realized. This could be a perfectionist’s dream but it would not necessarily prove a worth-pursuing exercise. The way ahead is probably one of knowing when and where to dissect liposomal fate and behaviour in the living animal. References

Gregoriadis, G. (1990), Immunological adjuvants: a role for liposomes. Imnunol. Toduy, 11, 89-97.

F. C. Szoka, Jr:

The chapters in this Forum reinforce the need for well-characterized liposomes if one is to dissect factors attributable to macrophage biology. For instance, Phillips (Forum) reminds us that water-soluble muramyldipeptides (MDP) rapidly leak from certain liposomes, making dose-response coriiparisons with non-encapsulated and lipophilic MDP compounds non-precise. If the compound is not in a liposome when it reaches the macrophage, interaction of the observed effect using the liposome paradigm may lead to an incorrect conclusion. I believe that the lack of carefully characterized liposome preparations, employed in some of the early work in the field, has created misconceptions on the liposome adjuvant effect as well in the modulation of macrophage tumoricidal activity. Our own experience in this area has shown that the method of preparation can dramatically affect the incorporation of amphipathic molecules such as lipopolysaccharide into liposomes. Wild-type LPS is poorly incorporated into multilameller vesicles prepared by the classical Bangham technique. High ef-

ficiency incorporation requires sonication combined with dehydration and rehydration (Dijkstra et al., 1988). We observed that when LPS is intercalated into the bilayer by the sonication-dehydrationrehydration protocol, even high-LPS density liposomes exhibit a decreased capacity to activate LAL coagulation (Dijkstra et al., 1988). The apparent contradiction in the data on LPS liposomes alluded to by Alving and co-workers (this Forum) might arise from comparing in vivo to in vitro experiments. One possibility to account for the difference is that in vivo, macrophages process the liposomal lipid A and regurgitate lipid A or fragments. The products of macrophage processing then activate B cells (or other cells) for antigen presentation but have little effect on the macrophages. The phenomena of low-dose LPS inducing tolerance to subsequent higher doses of LPS is well known. Liposomal LPS may in some fashion be mimicking this effect on the macrophages in the adjuvant experiments of Alving. Alternatively, the particular protocol used to prepare the liposomes might generate non-liposomal lipid A/LPS complexes that cannot be removed from MLV preparations by differential centrifugation or by separation on Sephadex G-75 columns (Dijkstra et al., 1988). Non-liposomal drug lipid complexes have arisen for the amphipathic compound amphotericin B when MLV preparations are used (Janoff et al., 1988). This may have unexpected benefits since the lipid-amphotericin B complex has less toxicity than simply intercalating the drug in liposomes (Janoff et al., 1988). Whether such an explanation applies in this case would require analysis of the malaria-lipid-A-liposomal adjuvant on a floatation gradient. Finally the results of Dumont and colleagues in table I (1990) cited by Alving and coworkers (Forum) in fact confirmed our finding that in vivo tumoricidal activity is eliminated when LPS is intercalated into liposomes; only in the case of mannosyl-targeted liposomes was the in vitro antitumour effect of LPS retained. Thus, we think that when LPS is intercalated into the bilayer it has a greatly reduced macrophage activation capability. Liposome-encapsulated muramyldipeptide compounds promoted for their anti-tumour effects through the activation of macrophages (see Daemen; Fidler; Phillips, this Forum) have advanced into a phase I clinical trial. In two out of three phase I clinical trials, peripheral blood monocyte tumoricidal activity (MTA) was variable and not correlated with dose (Urba et al., 1990; Creavens et al., 1990). In the third trial (Murray et al., 1990), a significant increase in MTA was found only at the intermediate dose and in patients that had a baseline cytotoxicity under 35 %. The assumption in the clinical studies is that the MDP-liposome formulation interacts with peripheral blood monocytes in vivo; however, evi-

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dence to support this assumption is surprisingly weak. We find (unpublished observation) that human peripheral blood monocytes do not avidly interact with liposomes, even negatively charged phosphatidylserine liposomes. Distribution studies of the technetium-labelled formulation indicated rapid clearance (circa 15 min T1/2) and localization in the RES. This suggests that tissue macrophages receive most of this formulation. If the monocyte is the target for liposome immunomodulators, it would seem advantageous to design a liposome that is targeted to circulating monocytes rather than a liposome that is rapidly cleared from circulation. Adverse effects observed in the MDP liposome trials exhibit similarities to those induced by low dose interleukin-I or tumour necrosis factor infusion (Dinarello, 1991). Perhaps surrogate markers for activation such as serum TNF levels may be a better indication of macrophage activation than MTA. The contributions by Phillips and Daemen focus on the tissue macrophage, particularly the Kupffer cell as a suitable target for activation by liposomal immunomodulators. The questions raised by Daemen and Phillips concerning the mechanism of action, duration of activation and accessibility of activated macrophages to the tumour cell are important. Phillips points out that a window of opportunity for macrophage killing exists as tumour cells transit the liver. This window may be quite important for successful macrophage activation therapy in an adjuvant setting to surgical removal of tumours, such as colon carcinoma. As answers to the questions posed by Daemen and Phillips become known, clinical protocols as well as liposomal formulations of macrophage activators, may have to be revised to maximize tumoricidal effects in humans. Other mysteries include why macrophages become refractory for activation and whether or not they can be reactivated for a second assault on tumour cells. Given the lifetime of Kupffer cells in the liver, reactivation may be necessary to maximize tumour cell killing. It seems the answers to many of the above questions await advances in macrophage cell biology; liposome-mediated oblation of macrophage activity could have a significant role here. Opportunities to use liposome macrophage suicidal techniques to address questions in macrophage defence and inflammatory responses are impressive (van Rooijen, this Forum). Liposomal delivery would be even more valuble as a tool if selective macrophage functions could be suppressed by encapsulated antisense or anti-gene molecules. In antigen presentation, the observations in this Forum (Huang et al; Harding et al.) that pH-sensitive liposomes can deliver antigens for class I presentation supports the idea that pH-sensitive liposomes can

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introduce macromolecules into the cytoplasm. Whether pH-sensitive liposomes are needed as vaccine formulations to elicit a more vigorous CTL response remains to be seen since Huang and coworkers (Forum) can also induce a strong CTL response with pH-stable liposomal antigen. Only a small fraction of the contents of cell-associated pHsensitive liposomes actually reach the cytoplasm ; the majority of the contents are conveyed to the lysosome where they are degraded (Chu et al., 1990). This may account for the large amount of pH-sensitive liposome antigen that must be added to the APC to observe class I presentation (Harding et al., this Forum). The use of various liposome types with differential pH stabilities (Harding et al., this Forum) is a clever way to explore compartments where antigens are degraded/interact with -MHC II molecules i.e. early versus late compartments. I would be interested in learning if a large dose of protein delivered in a lysosomally processed liposome can block a subsequent dose of antigen delivered in a pH-sensitive liposome. An appropriately designed experiment might distinguish between different pathways for antigen degradation/binding to MHC II. The results of Huang and co-workers (Forum) that both pH-sensitive and stable liposomes can induce a class I response in animals is another indication that the macrophage may be only the initial step in the processing of liposomal antigens in vivo. Future in vivo studies with liposome-encapsulated antigens and/or macrophage function suppressors may shed light on the cells and pathways involved in this process. Liposome techniques should continue to be useful tools in exploring this aspect of the immune response.

References

Chu, C-J., Dijkstra, J., Lai, M-Z., ef al. (1990), Efficiency of cytoplasmic delivery by pH-sensitive liposomes to cells in culture. Pharmaceut. Res., 7, 824834. Creavens, P. J., Cowens, J.W ., Brenner, D.E. et al. (1990), Initial clinical trial of the macrophage activator muramyltripeptide-phosphatidylethanolamine encapsulated in Iiposomes in patients with advanced cancer. J. biol. Resp. Mod, 9, 492498.

Dijkstra, J., Ryan, J.L. & Szoka, F.C. (1988), A procedure for the efficient incorporation of wild-type lipopolysaccharide into liposomes for use in immunological studies. J. Immunol. Merhods, 114, 197-205. Dinarello, C.A. (1991), The proinfammatory cytokines interleukin-1 and tumor necrosisi factor and treatment of the septic shock syndrome. J. infect. Dis., 163, 1177-l 184. Dumont, S., Muller, C.D., Schuber, F. & Bartholeyns, J. (1990), Antitumoral properties and reduced toxicity of LPS targeted to macrophages via normal or mannosylated liposomes. Anticancer Res.. 10, 155-160.

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Janoff, A., Boni, L.T., Popescu, M.C. et al. (1988), Unusual lipid structures selectively reduce the toxicity of amphotericin B. Proc. natl. Acad. Sci. (Wash.), 85, 6122-6126. Murray, J.L., Kleinerman, E.S., Cunningham, J.E. et al. (1989), Phase I trial of liposomal muramyl tripeptide phosphatidylethanolaine in cancer patients. J. C/in. Oncol., 7, 1915-1925. Urba, W.J., Hartman, L.C., Longo, D.L. et 01. (1990), Phase I and immunomodulatory study of a muramyl peptide, muramyl tripeptide phosphatidylethanolamine. Cancer Res., 50, 2979-2986.

C.V. Harding : We will comment primarily on issues raised by the other authors that pertain to antigen processing. As noted by F. Szoka, B cells do not efficiently process liposome-encapsulated antigens (LEA) while macrophages exhibit efficient processing, a finding that is confirmed by data obtained in our own laboratory. This deficiency appears to be explained by decreased uptake of LEA. It is unclear whether targeting of liposomes to B cells by receptor-mediated uptake could overcome this inherently inefficient liposome uptake and allow B cells to process LEA. Huang et al. report that acid-sensitive liposomes delivered antigen into the MHC-I processing pathway in EL-4 thymoma cells in vitro. This effect was not achieved using acid-insensitive liposomes. Their data are similar to the results we describe from our laboratory with peritoneal macrophages as antigenpresenting cells (although the specific, MHC-Irestricted T cell responses were measured differently: with a cytotoxicity assay in their studies and by T cell cytokine release in ours). In addition, preliminary data from our laboratory, like theirs, indicates that acid-sensitive liposomes injected in vivo can induce class I CTL responses (measured in vitro by a cytotoxicity assay), consistent with the in vitro studies. However, we also observed MHC-I-restricted CTL induction with acid-insensitive liposomes. This also parallels the observations of Huang et al., and differs from the results we have observed with these liposomes in vitro. Together, these data suggest that LEA can be processed and presented by pathways which have not been reconstituted in vitro. This processing may involve specialized APC or interactions among cells. The suggested role of dendritic cells in the presentation of LEA in vivo may be an important factor, but this warrants further scrutiny. Certainly, the discussions by Gregoriadis, Szoka, Claassen, Alving et al. and Van Rooijen highlight the central role of macrophages in the processing of LEA and materials. The hypothesis that peptides are “handed over” from

Y

macrophages to dendritic cells is interesting, but no clear mechanism for this is known and further study is required. In addition, the primary induction of OVA-specific CTL reported by Huang et al. involved dendritic cells (harvested 3 h after injection), but the induction was observed only with acid-sensitive liposomes. Thus, the in vivo presentation of antigen encapsulated in acid-insensitive liposomes (observed in their laboratory and in ours) was not recapitulated in this model. We conclude that the interactions of macrophages and dendritic cells and their exact roles in the induction of CTL responses in vivo remain unclear and require further study. However, the use of liposomes to deliver antigen as developed by Huang et al. promises to be a powerful tool for dissecting the MHC I pathway in vivo and in vitro. Other important approaches will include the targeting of antigen or the modulation or elimination of specific cell populations in vivo using the methods discussed by Gregoriadis, Claasen, Alving et al., Van Rooijen, Fidler, Daemen and Phillips.

L. Huang Harding

et al.

The conclusions of the work on the class-Imediated antigen presentation which is summarized in figure 1 basically agree with what we have found for the sensitization of the target cells (see Huang et al., this Forum). The hypothesis is interesting that antigen has to be delivered to the lysosome and the epitope is transported back to the endosome for the class-II-mediated presentation. I wonder if an alternative mechanism might also be consistent with their data. Suppose that the antigen processing as well as the complexation with the class II molecule all take place in an endosome compartment. Because the pHsensitive liposomes destabilize the endosome membrane, this mechanism would be impaired despite the fact that the antigen is also released in this compartment. Antigen delivered to the lysosome compartment by the pH-insensitive liposomes is largely degraded, and little would be salvaged and transported back to the endosome compartment. The amount of antigen actually presented might just be a small fraction of what is delivered to the lysosomes. Do any experiments dispute this alternative hypothesis? Also, I wonder what is the evidence for their repeated claim that only a small fraction of the antigen is released to the cytosol when the pH-sensitive liposomes are used to deliver the antigen. If so, the rest of the antigen should still come to the lysosomes and be processed for presentation.

LIPOSOMES

AND MACROPHAGE

Van Rooijen Several questions or comments have come to my mind after I read this interesting review. First, I wonder what cells in the liver are responsible for the uptake of liposomes after the Kupffer cells are eliminated by the liposomal Cl,MDP. Are these endothelial cells? What if they are also eliminated by a second injection of liposomal Cl,MDP? Second, systematic elimination of RES would lend an unprecedented opportunity to the study of extravasation of liposomes and other colloidal or macromolecular carriers. Any work done in this context? Third, it would be beneficial to selectively eliminate the macrophages of a given organ and leaving those in other organs intact. This could be performed by liposomes which are highly hepatotropic or those which are splenotropic. The splenotropic liposomes recently described by us could be useful for the latter (Liu et al., 1991). References Liu, D., Mori, A. & Huang, L. (1991), Large liposomes containing ganglioside GM accumulate effectively in spleen. Biochim. biopkys. Acta (Amst.), 1066,

159-165. Claassen What is described in the “Introduction” of the paper appears to be overly pessimistic about the immunoliposome targeting of drugs. The non-specific uptake by the RES can be suppressed by including the recently developed glycolipids or PEG-lipids in the liposome formulation (Maruyama et al., 1990). Instability and the fusion behavior of liposomes are also a function of the lipid composition and can be used to our advantage (see Wright and Huang, 1989). Undesirable uptake by cells with epitopes that are similar to or cross-reactive with the target antigen can be avoided or reduced by carefully selecting the appropriate targeting antibody. Inaccessibility of tumour or infected cells to the i.v. injected liposomes has been a real problem. But the recently developed long-circulating liposomes show increased accumulation of liposomes in the tumour mass (Gabizon and Papahadjopoulos, 1988), thus giving some hopeful solutions to the problem. Immunogenicity of the immunoliposome is a real problem which has no good solution at the moment. References Gabizon, A. & Papahadjopoulos, P. (1988), Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. nut. Acad. Sci. (Wash.), 85, 6949-6953.

FUNCTIONS

Maruyama, K., Kennel, S.J. & Huang, L. (1990), Liposome composition is important for highly efficient target binding and retention of immunoliposomes. Proc. nut. Acad. Sci. (Wash.), 81,5144-5148. Wright, S. & Huang, L. (1989), Antibody directed liposomesasdrug delivery vehicles.Advanc. Drug Delivery Rev., 3, 343-389.

Pate1 Gabizon and Papahadjopoulos (1988) and Allen et al. (1989) have proposed the “shielded negative charge” hypothesis for the structure feature of lipids which exhibit an activity to prolong the circulation time of liposomes. While the hypothesis is consistent with the structures of GM, and PI, our recent unpublished data raise some questions about its validity (Park and Huang, unpublished). We have modified the structure of GM, by first oxidizing the vicinal hydroxyl groups with periodate and then conjugating additional carboxylic groups by reductive amination with beta-alanine. When the modified GM, is incorporated into liposomes, the circulation time of the liposomes is about the same as those containing the native GM,. The added carboxylic groups locate on all saccharide residues including the terminal galactose, so these negative charges are not “shielded”. We have also found that N-glutaryl-PE and N-adipyl-PE have a considerable activity to prolong the circulation time of liposomes. Again, the negative charges of these lipids are not “shielded”. The mechanism of action of these lipids deserves further studies. References Gabizon, A. & Papahadjopoulos, P. (1988),Liposomeformulationswith prolonged circulation time in blood andenhanceduptakeby tumors.Proc. nut. Acad. Sci. (Wash.), 85, 6949-6953. Allen, T.M., Hansen,C. & Rutledge,J. (1989),Liposomes with prolonged circulation times: Factors affecting uptake by reticula endothelial and other tissues. Biochim. biophys. Acta. (Amst.), 981, 27-35.

C.R. Alving, J.N. Verma, M. Rao, U. Krzych, S. Amselem, S.J. Green and N.M. Wassef: Initial

interactions

of liposomes

with macrophages

Pate1 emphasizes that interaction of liposomes with plasma proteins causes attachment of opsonins that promote phagocytosis of liposomes by macrophages. As pointed out by him, our laboratory has proposed the concept that complement (C) activation by liposomes, mainly due to binding of naturally

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occurring anti-phospholipid or anti-cholesterol antibodies, may be a major factor that induces opsonophagocytosis of liposomes by macrophages. We have recently demonstrated that the so-called “stealth” liposomes (i.e. long-circulating liposomes containing ganglioside GM,, phosphatidylinositol or sulphatide), suppress the initiation of C-dependent phagocytosis of liposomes in vitro, and it is therefore our view that C activation may be an important factor in vivo that regulates the removal of circulating liposomes by macrophages (Wassef et al., 1991). In the development of liposomal vaccines it is important to remember that most vaccines are not administered to humans by intravenous or intraperitoneal injection. Vaccines are usually injected by the intramuscular route, and our experience has been that a substantial fraction of injected liposomes reside for a long period of time (probably for at least several weeks) at the i.m. injection site. The residence time of liposomes in the muscle therefore provides an opportunity for liposomes to interact with numerous types of cells that may be in the vicinity or may migrate to the injection site, including neutrophils, macrophages, lymphocytes and other cells. Inclusion of an adjuvant such as lipid A could allow the development of a prolonged local inflammatory reaction that could promote the secretion of cytokines and other factors that enhance the immune response. Activation

of macrophages by liposomal

lipid A

If the liposomes that are placed in a depot site have lipid A that is not visible on the surface (in our terminology, as explained in our contribution, if the liposomal lipid A is present in a low epitope density and the liposomes are “limulus-negative”), then it might be expected that the liposomal lipid A would be quiescent from a macrophage activation standpoint until it is phagocytosed by macrophages. In contrast, if the liposomal lipid A is in a high epitope density (“limulus-positive”), then the liposomal lipid A would be expected to have the capacity to activate macrophages by interacting with cell surface receptors even in the absence of phagocytosis. Since submitting our contribution to this Forum, we have had an opportunity to test the above hypothesis by comparing limulus-negative and limulus-positive liposomes for the ability to induce enhanced immunological presentation of a recombinant malaria antigen by macrophages. We have also examined the abilities of limulus-negative and limulus-positive liposomes to activate macrophages as determined by nitrite secretion. Our finding has been that when the total amount of lipid A added to the cells is held constant, limulus-negative and limulus-positive liposomes perform equally well for promoting enhanced antigen presentation and also for activating macrophages to secrete nitrite when

IN IikfMUNOLOGY compared with liposomes lacking lipid A. Based on this observation, we believe that our most recent data support the concept that macrophage activation by liposomal lipid A, at least for certain biological activities, can be initiated from an intracellular site. Daemen reviews evidence that gamma-interferon (IFNy) activates macrophages by interacting with receptors on plasma membrane, but that a second signal (endotoxin or MDP) is required to cause the macrophages to be tumoricidal. She points out that most of the target sites for MDP in macrophages are present in intracellular locations. She also reviews her previously described evidence that liposomal LPS or lipid A has a reduced ability to activate macrophages to tumoricidal activity, and she reiterates the hypothesis that liposomal lipid A activates macrophages only after binding to receptors on the plasma membrane. As we outlined in our contribution, this hypothesis could very well be correct for tumoritidal activation and is part of a controversy that still stands. We proposed, and we still believe, that the comparison of limulus-negative and limulus-positive liposomes might be a useful technique for examining the possible binding of liposomal lipid A to receptors on the plasma membrane that might be responsible for activation of macrophages for tumoricidal activity. However, as we have outlined above, we now have new experimental evidence that the activation of macrophages to secrete nitrite and to cause increased immunological presentation of a liposomal protein antigen can be initiated from an intracellular site after phagocytosis of the liposomes. Intracellular

fate of liposomal

antigens

Several authors (Szoka; Huang et al.; Harding et al.) emphasize the importance of understanding the intracellular fate of liposomal antigens in studies on the immunological presentation of the antigens by macrophages. We have recently provided evidence that at least a fraction of phagocytosed liposomal malaria antigen apparently escapes from the phagocytic or lysosomal vacuoles of macrophages and enters the cytosol where it can be easily detected as free antigen by immunogold electron microscopy (Verma et al., 1991). These observations suggest that liposomal antigen that is present in ordinary MLV liposomes can be delivered to the cytosol, and this could have implications for theories relating to the mechanism of presentation of liposomal antigen by macrophages. References Verma, J.N., Wassef, N.M., Wirtz, R.A., Atkinson, C.T., Aikawa, M., Loomis, L. & Alving, C.R. (1991), Phagocytosis of liposomes by macrophages : intracel-

LIPOSOMES

AND MACROPHAGE

lular fate of liposomal malaria antigen. Biochim. biophys. Acfu (Amst.), 1066, 229-238. Wassef, N.M., Matyas, G.R. & Alving, C.R. (1991), Complement-dependent phagocytosis of liposomes by macrophages: suppressiveeffects of “stealth” lipids. Biochem. biophys. Res. Commun., 176, 866-874.

I.J. Fidler:

Liposomes as (G. Gregoriadis)

immunological

adjuvants

Traditionally, immunization of mice has been carried out by intradermal administrations of antigens admixed with one or another adjuvant. The availability of liposomes containing both antigens and an adjuvant suggests exploring intravenous administration. Liposomes consisting of phosphatidylcholine localize to organs rich in RES activity (liver, spleen, lymph nodes). Generation of immunity (humoral and cellular) subsequent to intravenous administration should be compared to that after intradermal injection. Similarly, intralymphatic administration of liposomes containing antigens and an adjuvant could enhance beneficial response. Macrophage

activation

(7: Daemen)

Dr. Daemen is correct in stating that many questions regarding the mechanisms of macrophage activation remain unanswered. However, several aspects of this process are clear. Immunomodulators can bind either to all surface receptors (IFNy) or to intracytoplasmic receptors (MDP). Signal transduction pathways and phosphorylation by protein kinase C or protein tyrosine kinases are involved in the process of activation, At least for IFNy, it is now becoming apparent that, once internalized, this biological molecule leads to the generation of a secondary DNA-binding protein which in turn leads to production of mRNA and final products.

N.C. PhiI~ips:

The papers presented in this Forum cover part of the ever increasing spectrum of potential liposomal applications. The papers fall naturally into 3 areas : macrophage activation against tumour cells, vaccine activity and liposomes as tools to study macrophage function. In the present discussion I would like to highlight some of the data presented in the context of my own research programmes.

FUNCTlONS

Macrophage

tumoricidal

251

activity

The paper by Fidler describing systemic macrophage activation and subsequent tumour cell killing in vitro and in vivo by a variety of liposomal encapsulated immunomodulatory agents, especially those based on lipophilic muramyl dipeptides (MDP) exemplifies the potential of liposomal therapy in oncology. Based on our experimental and clinical phase I data from the use of liposomal MDP in the treatment of liver metastases, I would, however, like to introduce a cautionary note concerning the unsubstantiated generalization of the applicability of this therapeutic approach. Tumour cells and monocytes are both subject to rapidly changing physical locations. It was fortunate that the initial studies where liposomal immunomodulator treatment was shown to be effective, namely eradication of lung metastases, utilized a model where concomitant tumour cell metastasis and monocyte/macrophage migration occurred. This is certainly not the case for the liver, an organ that clinically is the major site of tumour metastasis. The results presented in the paper by Daemen, as well as our own results, amply demonstrate the effectiveness of liposomal immunomodulators for activating Kupffer cells, the tissue-fixed macrophage of the liver, to a tumoricidal state. Tumour cell burden, as proposed by Daemen, may well be a limiting factor for liposomal immunomodulator therapy. It is my contention, however, that the major factor that determines whether such therapy will be effective is the time of liposomal therapy and the potential contact between metastasizing tumour cells and nonmigratory Kupffer cells. Kupffer cells do not migrate into the parenchyme of the liver towards tumour cell foci, irrespective of whether or not they have been treated with liposomal immunomodulators. Our results indicate that tumour cells that metastasize rapidly from the liver sinusoids to the parenchyme are the least susceptible to Kupffer cell-mediated defence mechanisms, and that in situ induced parenchymal primary hepatocellular tumours are totally unaffected by liposomal immunomodulators. Our phase I studies with liposomal lipophilic MDP in patients with retinal melanoma who had measurable hepatic metastases confirm the limitation of this approach, in that significant activation of circulating monocles occurs in the absence of any inhibitory effect on liver tumours (N.C. Phillips and A. Loutfi, unpublished data). The presence of liver micrometastases at the initial diagnosis of a significant proportion of primary tumours (for example primary colorectal carcinoma or retinal melanoma) makes the impact of liposomal immunomodulator treatment very uncertain. Liposomal therapy of individuals with preexisting hepatic micrometastases may well be a case of “closing the stable door after the horses have bolted”.

252 Liposomal

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IN IMMUNOLOGY

vaccines

The papers presented by Alving, Gregoriadis, Harding, Huang and Szoka emphasize the potential of liposomes to act as immunoadjuvants for associated antigens. While a consensus regarding the role of the macrophage as an antigen-processing cell (APC) is evident, the effectiveness of passive or active targeting of liposomal antigens to other APC such as Langerhans cells, dendritic cells or B cells remains relatively obscure. Data presented in the papers by Harding and Huang do, however, indicate some relationship between macrophages and dendritic cells. The possibility of organ-specific liposomal localization via the use of tissue-specific opsonins in the paper presented by Pate1 may help resolve this question. The potential for utilizing liposomes to enhance MHC-Vpeptide interaction urgently requires further development, especially for immunosuppressive retroviral antigens. Based on my experience with liposomal tumour antigens as vaccines in preclinical and clinical studies (Phillips, N.C. et al., 1990, J. Biol. Resp. Modif., 6, 58 1-6, 1987 ; Phillips, N.C., Prog. clin. biol. Res., 1990, 354A, 257-70), and on the modulation of immune responses in normal and retrovirus-immunosuppressed mice by liposomal adjuvants (Phillips, N.C. and Emili, A., Vaccine, 1991, in press; Phillips, N.C., submitted), I would like to offer some additional comments concerning the role of the liposome itself, the use of additional immunoadjuvants incorporated within the liposome and the effect of liposomal adjuvants on IgG-isotype antibody responses. Liposomal phospholipid apparently does not affect the processing of soluble protein antigen (Harding). However, phospholipids enhance peptide/MHC-II interactions (Roof, R.W. ef al., Proc. nat. Acad. Sci. (Wash.), 1990, 87, 1735-39), making it likely that the liposome itself will influence the subsequent immune response. The stability of the liposome may be a secondary factor to its toxicity towards macrophage APC. Prior to initiation of immunization studies with liposomal antigens, my group carried out a number of studies to determine the toxicity of a range of liposomal preparations towards macrophages. Liposomes containing phosphatidylserine or phosphatidylethanolamine exhibit considerable toxicity in the range 0.1-I umol/ml, concentrations that are readily achievable during S.C. or i.m. injections. The paper by Alving et al. describing the effect of liposomes containing lipid A illustrates the potential for the concomitant incorporation of a macrophage-activating agent in liposomal adjuvants. The incorporation of lipophilic MDP, a potent macrophage activator, within liposomes is also effective in enhancing cellular and humoral responses against liposomal antigens, and results in even greater antibody levels (Phillips, N.C. et al. (1988), Cancer Detect. Prevent., 12, 451-59; Phillips, N.C. and Emi-

li, A. (1991), Vaccine (in press)). The possibility that enhanced induction of MHC-II or enhanced lysosomal processing in macrophage APC by such agents plays a role in enhancing the immune response requires elucidation. The potential for the generation of new immunogenic epitopes as consequence of macrophage activation has to be addressed. The paper by Gregoriadis delineates the role of liposomes as immunoadjuvants. Insufficient attention has however been paid to the effect of liposomal immunoadjuvants in inducing antibodies of all IgG isotypes, rather than, for example, IgGl antibodies induced by adjuvants such as alum. Experimental data from my group has shown that liposomes are significantly more potent as adjuvants in mice (5-lo-fold) than MDP or alum. Immunization protocols using minimal effective doses of a number of protein antigens show that in contrast to alum or MDP, where the IgG antibody is > 95 % IgGl, liposomes induce IgGl (50 (J~o),IgG2a/2b (40 To) and IgG3 (10 Vo). Furthermore, the IgG2a/IgG2b antibody ratio is strain-specific, with a predominance of IgG2a in A mice and a predominance of IgG2b in C57BL/6 mice. Both of these isotypes are capable of participating in ADCC reactions, and the ability of liposomal adjuvants to elicit these antibody isotypes represents a significant and major advantage over other immunoadjuvants. Liposomes

as tools to study macrophage function

The papers presented by Bogers, Claassen, Derijk, van Lent and van Rooijen illustrate the potential for characterizing macrophage function via the use of liposomes containing diphosphonates. The observation that elimination of tissue-fixed macrophages results in the unmasking of endothelial cell phagocytosis (Bogers et al.) or of immunosuppressive macrophage populations (van Rooijen) presents a number of entirely new vistas for liposome drug targeting. Macrophage/endothelial cell functions tend to be complementary, and the use of liposomal diphosphonates provides an elegant tool with which to investigate the relationship of these two cell types in, for example, neutrophil migration towards inflammatory sites or of tumour cell metastasis from the haematogenous compartment.

T. Daemen: The technique described by Eric Claassen on the use of dioctadecyl-tetramethylindocarbocyanine (DiI) for the labelling of membranes has recently been applied by us, In our study, in vitro labelled rat monocytes, after intravenous injection, could be traced

LIPOSOMES

AND MACROPHAGE

back by fluorescence microscopy in liver, spleen and lungs. This method offers significant advantages over other methods, especially for the detection of low numbers of cells. Another advantage of this technique is that cells can be characterized immunohistochemically subsequent to photoconversion of the DiI. The solution of the controversy regarding macrophage activation with liposomal lipid A, as proposed by Alving et al., can, in my opinion, not be made based on the studies by Fogler et al. (1983) and Dumont et al. (1990). The former used wild-type LPS from Escherichia coli 055:B5 and estimated the amount of LPS in the liposomes on the basis of entrapped aqueous volume. However, LPS can become associated with liposomes in various ways, including the association with the liposomal bilayers. In the studies described by Dumont et al., wildtype LPS from Salmonella abortus equi is used, encapsulated in REV. These authors did not find any in vitro activation of monocyte-derived macrophages with LPS-liposomes ; only a minor effect with mannosylated LPS-liposomes was found. The lack of activation with their LPS-liposomes is surprising, since the method they used for the removal of nonencapsulated LPS from the liposomes, i.e. gelfiltration on Sephadex-G75, is not effective. Thus, the lack of activation with 10 ug/ml of liposomal LPS, which is 1,OOO-fold the concentration we need for the activation of liver macrophages, is presumably due to the very high level of cytotoxicity displayed by control macrophages in this study (50 070). The preparation of LPS-liposomes by the methods described by Dijkstra et al. (1987, 1988a) results in more than 99 % incorporation of LPS in the liposomal bilayers. Experiments performed with multilamellar vesicles composed of eggPC, eggPG and cholesterol (4/4/l molar ratio) show that even with vesicles containing 40 to 100 pg of Re-LPS per pmol of phospholipid, macrophage-mediated tumourcytotoxicity, cytokine release and stimulation of murine splenocyte proliferation was reduced 100 to 1,OOO-fold when compared to free LPS (Dijkstra, 1987, 1988a, 1988b, 1991 in press). The methods employed to prepare LPScontaining liposomes and the procedures used to remove non-entrapped material, most likely determine the potency of the liposome-associated LPS. If LPS is not adequately inserted or encapsulated but, rather, adsorbed to the liposomal membrane, the lipid A moiety can still be accessible for cell-associated molecules involved in the activation mechanism. References Fogler, W.E.,

Talmadge, J.E. & Fidler, I.J. (1983),

J. Reticuloendoth. Sot., 33, 165.

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253

Dumont, S., Muller, C.D., Schuber, F. & Bartholeyns, J. (1990), Anticancer Res., 10, 155. Dijkstra, J., Mellors, J.W., Ryan, J.L. 8~ Szoka, F.C. (1987), J. Immunol., 138, 2663. Dijkstra, J., Ryan, J.L. & Szoka, F.C. (1988a), J. Immunol. Methods. 114, 197. Dijkstra, J., Larrick, J.W., Ryan, J.L. & Szoka, F.C. (1988b), J. Leuk. Biol., 43, 436. Dijkstra, J. & Ryan, J.L. (1991), in “Bacterial endotoxic lipopolysaccharides”, vol. 1 (Morrison, D.C. &Ryan, J.L.). CRC Press, Boca Raton, FL (in press).

W. Bogers: The papers included in this Forum illustrate the use of liposomes in a very broad field. Most of the contributors have described the use of liposomes to target particular molecules selectively to macrophages. Using this approach, liposomes can be used to target antigens, immunomodulators or cytotoxic drugs (like Cl,MDP) to macrophages. The main goal of using cytotoxic drugs, encapsulated in liposomes as described in this issue, is studying macrophage function(s) by elimination of macrophages in vivo. In this respect we want to add another aspect. After elimination of macrophages it is possible to study (unknown) functions of other cell populations in vivo which normally don’t come “to the surface” because of the presence of macrophages. An example has been described in our contribution : the clearance and handling of IgA and IgG aggregates appeared to be mediated by liver endothelial cells after elimination of liver and spleen macrophages. Whether this process can take place only after macrophage elimination or also in normal situations, is not clear at present. One of the problems in studying the function(s) of other cell populations in vivo, after elimination of macrophages, is that (important or essential) interactions between macrophages and other cells may be disturbed, possibly inducing nonphysiological processes.

R.H.

Derijk

and F. Berkenbosch:

Bogers et al. The authors of this paper show elegantly that a well-defined function of macrophages (Kupffer cells), being clearance of immune aggregates, is rapidly compensated by liver sinusoidal endothelial cells in the absence of macrophages. This example clearly shows that a lack of change in complex function (e.g. clearance of immune complexes) after macrophage depletion does not necessary allow the conclusion

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that macrophages do not participate in such complex function. Conversely, also false-positive conclusions can easily be obtained about the role of macrophagelike cells in a complex function utilizing the liposomemediated macrophage depletion. During the time of absence of macrophages, changes may occur in function and responsivity of immune and accessory cells due to a lack of regulatory signals normally provided by macrophages. Therefore, a deficit in complex functions in macrophage-depleted rats does not necessary indicate that macrophage are directly involved in the complex function studied. P.L.E.M.

van Lent et al.

The present study is in line with other studies showing that depletion of macrophages is a useful tool in attempting to understand the in vivo role of macrophages in a variety of immune responses. However, care should be taken by choosing an appropriate experimental model to study the role of macrophages by the liposome-mediated macrophagesuicide technique in autoimmune diseases. In the case of rheumatoid arthritis, the used models are based on the induction of a local inflammation by injecting antigens in the joint cavity. Antigens, especially in opsonized forms, will activate cells of the macrophage lineage. In turn, these will release cytokines and other proinflammatory factors attracting and activating other immune cells like T cells and granulocytes, leading to a local and systemic immune response. The processes involved are very similar to any ordinary inflammation and are likely not to reflect the initiating processes occurring in human rheumatoid arthritis or other autoimmune diseases. One of the hall-marks of organ-specific autoimmune diseases including arthritis is the presence of a disbalance between effector and suppressor T cells, combined with a disturbed and/or elevated self-antigen expression. In case of autoimmune arthritis, the first effector cells to be initiated are likely to be autoreactive T cells [ 11. Recently, we have reviewed evidence that in a variety of spontaneous forms of animal autoimmunity, macrophages are relatively unresponsive to endotoxin in the synthesis/release of immune regulatory soluble products, such as the cytokines interleukin-1 (ILl) and tumor necrosis factor (TNF) [2]. Interestingly, repeated treatment of such animals, prior to the onset of the autoimmunity with small amounts of ILI, TNF, endotoxin or viral particles will largely inhibit the onset of the disease, presumably by upregulating deficient macrophage functions (Derijk and Berkenbosch, unpublished). From these studies, we have concluded that a macrophage dysfunction, either genetically or environmentally induced, can be an essential cofactor that contributes to the development/onset of some autoimmune diseases. Our conclusion is reinforced by the recent data

of Cornall et al. [3], showing that in the case of type I diabetes in mice, the susceptibility of the disease is genetically linked to IL1 receptor and Lsh/It4/Bc9 genes. It is worth noting in this respect that the Lsh/It4/B9c gene, which has yet to be cloned, is known to influence the activity of macrophages. In summary, the data of Lent et al. obtained by their arthritis model suggest that macrophage-like cells are involved in the onset of arthritis and therefore should be suppressed or eliminated. In contrast, the data briefly discussed above may favor the view that methods should be used to enhance rather than to suppress macrophage functions to control the onset of some forms of autoimmune diseases. Whether such methods are also useful to suppress the onset of human rheumatoid arthritis, may be subject to further study.

C.R. Alving,

G. Gregoriadis

and F.C. Szoka, Jr.

The presented studies point towards the beneficial effects of potentiating the macrophage functions by methods involving liposomes in order to create more efficient vaccines. In general, these studies are primarily focused on enhancing the antigenpresenting capacity of the macrophage but they bypass the issue of the important role of macrophage activation in orchestration of the total host response. As reviewed in this issue, we have shown that activation of macrophages results in release of factors that lead to changes in neuroendocrine functions such as for instance increased pituitary-adrenal activity. The latter results in sustained increased plasma corticosterone levels. Evidence is accumulating that this response may determine the specificity as well as the magnitude of the immune response. Gilles et al. [4] have demonstrated that resting immune cells are much more sensitive for the inhibiting actions of glucocorticoids than activated cells. This observation initiated the view that glucocorticoids may function to downregulate excessive expansion of cells with low affinity for the antigens and allow progressive expansion of cells with high affinity for the antigen. Thus, this mechanisms will greatly facilitate the specificity of the immune response to a particular antigen. Moreover, glucocorticoids may even potentiate the proliferation and/or function of these committed cells with high affinity to the particular antigen, thereby enhancing the magnitude of the specific immune response. This view is based on observation showing that glucocorticoids enhance cytokine receptor expression, potentiate the effects of IL1 and IL6 on B-lymphocyte function and augment the primary humoral response to antigens. Thus, creating improved vaccines by liposome targeting may not only involve the boosting of the antigen-presenting capacity of macrophages, but also

LIPOSOMES

AND MACROPHAGE

may involve other macrophage-coordinated responses such as discussed here for the activation of the pituitary-adrenal system. References [ 1] W. Van Eden et al. (1989), A cartilage-mimicking T-cell epitope on 65K mycobacterial heat-shock protein : adjuvant arthritis as a model for human rheumatoid arthritis. C. T.M.I., 145, 27-43. [2] Derijk, R.H. & Berkenbosch, F. (1991), The immunehypothalamo-adrenal axis and autoimmunity. Intern. J. Neuroscience, 59, 91-100. [3] Cornall, R.J. et al. (1991), Type I diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1. Nature (Lond.). 353,

262-264. [4] Gilles, S., Crabtree, G.R. & Smith, K.A. (1979), Glucocorticoid-induced inhibition of T-cell growth factor production. J. Immunol., 123, 1624-1631. [5] Tuchinda, M., Newcomb, R.W. & Devald, B.L. (1972), Effect of prednisolone treatment on the human immune response to keyhole limpet hemocyanin. Znt.

Arch. Allergy., 42, 533-544. [6] Emilie, D. et al. (1988), Glucocorticoid-dependent synergy between interleukin-1 and interleukin-6 for human B-lymphocyte differentiation. Europ. J. Immunol., 18, 2043-2047.

E. Claassen :

The role of heterogeneous macrophage subsets in liposome targeting and immunomodulation This Forum reinforces the notion that liposomes are useful tools in answering a number of biological questions. Almost any compound can be, or has been, associated with liposomes and used in vitro or in vivo, albeit with variations in efficacy. These differences could be due to technical differences (e.g. epitope density, lipid composition, liposome size, concentration of solute etc.), as described in this Forum, or to the involvement of different types of macrophages. In view of the fact that it is still fairly common - see also this Forum - to talk about “the macrophage” I would like to illustrate my point that “the macrophage” does not exist. Inter-population heterogeneity. - When administering liposomes intraperitoneally one can reach peritoneal, splenic and liver macrophages. However, after intravenous administration of lipid vesicles, peritoneal macrophages will not come into contact with the liposomes. After subcutaneous administration (of modest doses), liposomes will reach lymph node macrophages only. This is of particular importance since macrophage populations have

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different functions. This is reflected, for example, in the fact that macrophages from lymphoid tissues can usually present antigens to the immune system, whereas for instance alveolar and in some cases even peritoneal macrophages have been shown to suppress antigen presentation in vivo and in vitro, in certain models (Thepen et al., 1989; W.M. Kast, AZL Leiden, pers. comm.). When liposomes are taken up by non-antigen-presenting macrophages they are sequestered from the immune system. Presentation of liposomal antigens is therefore a subtle equilibrium between availability of liposomes for antigenpresenting cells/macrophages and clearance of liposomes by non-presenting macrophages (including Kupffer cells which are poor antigen presenters and class-II-negative in e.g. mice). The non-presenting cells can be recognized because they are usually negative (or low) for MHC class II markers and do not produce interleukin-1 (Kurt-Jones et al., 1986). Intra-population heterogeneity. - In addition to the effect that routing of liposomes will have on the particular macrophage population that is primarily involved, there is also the matter of macrophage subsets within the different populations. In the spleen alone, over 11 different types of macrophages have already been identified (Leenen, 1989). Each subset is characterized by different surface markers and possesses (as far as known) different functions. In addition to this, at least four different subpopulations have been described among peritoneal macrophages (Beelen and Walker, 1983). In the blood, monocytes can be separated into distinct subpopulations with different activities in tumour-cell killing or accessory function for B or T cells (Figdor et al., 1982). Therefore it is easy to realize that activation, suppression or elimination of all macrophages (or even elimination of specific populations) as part of an experimental approach will not necessarily provide an unequivocal answer. To illustrate my point, the role of marginal zone macrophages in the immune response against thymus-independent type-2 (TI-2) antigens is of importance. Due to the fact that TI-2 antigens are selectively taken up and retained for long periods of time by these macrophages, numerous authors concluded that they would be involved in presentation of these antigens to the immune system, although it was known that they were MHC-IInegative. These ideas were confirmed by splenectomy studies and studies in which all cells from the splenic marginal zone were eliminated, in which a drastic reduction of the anti-TI-2 response was observed. However, in studies with an immunotoxin or liposomes which specifically suppressed marginal zone macrophages, we were not able to demonstrate a role for these cells in the TI-2 response. It was not until recently that we could demonstrate that marginal zone and follicular B cells were responsible for induction of a TI-2 response. Furthermore, we demonstrated that marginal zone macrophages ac-

41.91FORUM

IN IMMUNOLOGY

tually had a profound negative effect on the TI-2 response by obscuring the antigen from the immune system (see E. Claassen, this Forum). Our earlier results could easily be explained by indirect effects resulting from macrophage elimination, i.e. the release of proteolytic enzymes by dying macrophages resulting in severe damage to, and death of, adjacent B cells. Other indirect effects, resulting from macrophage modulation, have already been mentioned as the major hurdle for correct interpretation of experimental data by other authors in this Forum. Another important fact to take into account is the significant differences that are found in different species (Claassen, 1991) especially with regard to MHC class II expression of a given specific subset (e.g. alveolar macrophages ; class-II-positive in activated human lung, low in rats, negative in mice). Apart from the fact that macrophages are a very heterogenous population of cells with extreme differences in phenotype and function, one should also realize that some (if not all) macrophage functions can also be performed by other cell types and mechanisms. Some of these alternatives can even be better than the macrophage analogue, e.g., dendritic cells can present protein antigens up to lOO-fold better than antigen-presenting macrophages. In conclusion, I feel that, although the results shown in this Forum demonstrate great promise in the use of liposomes for macrophage modulation, great care should be taken with the interpretation of the results and their general applicability. One of the aspects which definitely needs further research is the role of macrophage subsets. The results of such studies could then lead to specific and improved targeting of liposomes to the subset which can most effectively function as effector cell to render the desired biological effect. References (additional Forum)

to those already listed in the

Beelen, R.H.J. & Walker, W.S. (1983), Dynamics of cytochemically distinct subpopulations of macrophages in elicited rat peritoneal exudates. Cell. Immunol., 82, 246-257. Claassen, E. (1991), Histological organization of the spleen : implications for immune functions in different species, in 38th Forum in Immunology”. Res. Immunol., 142, 313-372. Figdor, C.G., Bont, W.S., Touw, I., De Roos, J., Roosnek, E.E. & De Vries, J.E. (1982), Isolation of functionally different human monocytes by counterflow centrifugation elutriation. Blood, 60, 46-53. Kurt-Jones, E.A., Virgin, H.W. & Unanue, E.R. (1986),

Key-words: Liposome, in, MDP, Autoimmunity,

In vivo and in vifro expression of macrophage membrane interleukin-1 in response to soluble and particulate stimuli. J. Immunol., 137, 10-14. Leenen, P.J.M. (1989), Phenotypical analysis of murine macrophage differentiation. Thesis Erasmus University, Rotterdam, The Netherlands. ISBN 90-9003 179-o.

H. Patel: The papers included in this Forum illustrate how liposomes can serve as excellent carriers for biological response modifiers and immunomodulating agents that affect macrophage functions. It is both exciting and interesting that the techniques of liposome-mediated activation of macrophages initially reported by Fidler and his colleagues and elimination of macrophages demonstrated by van Rooijen and co-workers, are now exploited for further understanding of the functional role of macrophages in various biological processes and particularly in the host defence against infection and cancer. It is now clear that immunomodulator entrapped liposomes therapy is effective in eradicating both lung and liver metastases provided the bulk of tumour burden is first reduced by using conventional treatments. Next, investigation in the mechanism of PSmediated macrophage recognization of neoplastic cells should lead us to the development of liposomes which can be selectively targeted to tumour cells in the RE organs. It will be interesting to find out whether the activated macrophage can eradicate bacterial infection in cystic fibrosis patients. Infection in these patients is often the cause of death. The persistence of P. aeruginosa infections in cystic fibrosis patients and in other chronic airways diseases is believed to be caused by the dysopsonic activity of the polypeptide fragments generated from IgG by the pathogen-derived elastase which inhibits phagocyte/bacteria adherence and phagocytosis (Bainbridge and Fick, 1989). Aerosol delivery of liposomes with immunomodulator may activate macrophages which could perhaps over come the problem of dysopsonin and eradicate infection in these patients. References Bainbridge, T. & Fick, R.D. (1989), Functional importance of cystic fibrosis immunoglobulin G fragments generated by Pseudomonas aeruginosa elastase. J. Lab. Clin. Med., 114, 728-135.

Macrophage, Tumour, Lymphocyte, Lipids, Immunotherapy; Forum.

Immunoregulation;

MHC,

Liver, Endotox-