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Background: There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved., Methods: The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation., Results: The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia., Conclusions: Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.

Background: Obesity-related adipose inflammation has been thought to be a causal factor for the development of insulin resistance and type 2 diabetes. Infiltrated macrophages in adipose tissue of obese animals and humans are an important source for inflammatory cytokines. Clodronate liposomes can ablate macrophages by inducing apoptosis. In this study, we aim to determine whether peritoneal injection of clodronate liposomes has any beneficial effect on systemic glucose homeostasis/insulin sensitivity and whether macrophage content in visceral adipose tissue will be reduced in diet-induced obese (DIO) mice., Methodology/principal Findings: Clodronate liposomes were used to deplete macrophages in lean and DIO mice. Macrophage content in visceral adipose tissue, metabolic parameters, glucose and insulin tolerance, adipose and liver histology, adipokine and cytokine production were examined. Hyperinsulinemic-euglycemic clamp study was also performed to assess systemic insulin sensitivity. Peritoneal injection of clodronate liposomes significantly reduced blood glucose and insulin levels in DIO mice. Systemic glucose tolerance and insulin sensitivity were mildly improved in both lean and DIO mice treated with clodronate liposomes by intraperitoneal (i.p.) injection. Hepatosteatosis was dramatically alleviated and suppression of hepatic glucose output was markedly increased in DIO mice treated with clodronate liposomes. Macrophage content in visceral adipose tissue of DIO mice was effectively decreased without affecting subcutaneous adipose tissue. Interestingly, levels of insulin sensitizing hormone adiponectin, including the high molecular weight form, were significantly elevated in circulation., Conclusions/significance: Intraperitoneal injection of clodronate liposomes reduces visceral adipose tissue macrophages, improves systemic glucose homeostasis and insulin sensitivity in DIO mice, which can be partially attributable to increased adiponectin levels.

A liposome mediated macrophage "suicide" approach has been developed, based on the liposome mediated internalization of the small hydrophilic molecule clodronate in macrophages J Leukoc Biol 62:702, 1997. This molecule has a very short half life when released in the circulation, but does not easily cross phospholipid bilayers of liposomes or cell membranes. As a consequence, once ingested by a macrophage in a liposome encapsulated form, it will be accumulated within the cell as soon as the liposomes are digested with the help of its lysosomal phospholipases. At a certain intracellular clodronate concentration, the macrophage is eliminated by apoptosis. Given the fact that, neither the liposomal phospholipids chosen, nor clodronate are toxic to other (non-phagocytic) cells, this method has proven its efficacy and specificity for depletion of macrophage subsets in various organs. In several cases, organ specific depletion can be obtained by choosing the right administration route for the clodronate liposomes.

Liposomes have proven their use as a tool in various immunological studies. In our own studies, both their application as antigen carriers and as drug carriers appeared to be useful. Immune responses were elicited against free soluble protein antigens and against the same antigens in a liposome-associated (particulate) form, in order to compare both types of response. Since we were especially interested in the role of splenic macrophages in both types of response, we developed a liposome-mediated macrophage suicide approach, based on the liposome-mediated internalization of the small hydrophilic molecule clodronate in macrophages. This molecule has a very short half life when released in the circulation, but does not easily cross phospholipid bilayers of liposomes or cell membranes. As a consequence, once ingested by a macrophage in a liposome-encapsulated form, it will be accumulated within the cell as soon as the liposomes are digested with the help of its lysosomal phospholipases. At a certain intracellular clodronate concentration, the macrophage is eliminated by apoptosis. Given the fact that neither the liposomal phospholipids chosen nor clodronate are toxic to other (non-phagocytic) cells, this method has proven its efficacy for depletion of macrophage subsets in various organs. In several cases, organ-specific depletion can be obtained by choosing the right administration route for the clodronate liposomes.

Liposomes encapsulating the bisphosphonate clodronate can be used for the transient suppression of macrophage functions. Given the important role of macrophages in various disorders, the application of clodronate liposomes has been studied in several models of rheumatoid arthritis, neurological disorders such as experimental allergic encephalomyelitis and spinal cord injury, autoantibody mediated disorders such as immune thrombocytopenic purpura (ITP) and for the improvement of the efficacy of gene transfer and drug targeting.

This study describes the effect of the lipid dose of (99m)Tc-polyethylene glycol (PEG) liposomes in the low-dose range (0. 02-1.0 micromol/kg) on the pharmacokinetics and biodistribution in rats, rabbits, and humans. The biodistribution and pharmacokinetics of (99m)Tc-PEG liposomes at various dose levels were studied in rats and rabbits with a focal Escherichia coli infection. Scintigraphic images were recorded on a gamma camera. In addition, the role of macrophages in the biodistribution of a low-dose PEG liposome injection was studied. Finally, the pharmacokinetics of (99m)Tc-PEG liposomes at two lipid dose levels was studied in four patients. At a dose level of 0.03 micromol/kg, the blood level in rats at 4 h postinjection was significantly lower than at the highest dose level (1.1 micromol/kg). The same effect was observed in rabbits where enhanced clearance was observed at a dose level of 0.02 micromol/kg. The circulatory half-life decreased from 10.4 to 3.5 h (at 1.0 and 0. 02 micromol/kg, respectively). At the lowest dose level, liposomes were mainly taken up by the liver and to a lesser extent by the spleen. Injection of a low dose of PEG liposomes in macrophage-depleted rabbits resulted in normal pharmacokinetics, suggesting involvement of macrophages in the effectuation of the rapid elimination of the liposomes from the circulation. Most importantly, the rapid clearance of low-dose PEG liposomes was also observed in humans when relatively low lipid doses were administered. This study showed that at very low lipid doses the biodistribution of PEG liposomes is dramatically altered.

Quinolinic acid is a neurotoxic tryptophan metabolite produced locally during immune activation. The present study tested the hypothesis that macrophages are an important source. In normal gerbils, the macrophage toxin liposome-encapsulated clodronate depleted blood monocytes and decreased quinolinic acid levels in liver (85%), duodenum (33%), and spleen (51%) but not serum or brain. In a model of CNS inflammation (an intrastriatal injection of 5 microg of lipopolysaccharide), striatal quinolinic acid levels were markedly elevated on day 4 after lipopolysaccharide in conjunction with infiltration with macrophages (lectin stain). Liposome-encapsulated clodronate given 1 day before intrastriatal lipopolysaccharide markedly reduced parenchymal macrophage invasion in response to lipopolysaccharide infusion and attenuated the increases in brain quinolinic acid (by 60%). A systemic injection of lipopolysaccharide (450 microg/kg) increased blood (by 38-fold), lung (34-fold), liver (23-fold), spleen (8-fold), and striatum (25-fold) quinolinic acid concentrations after 1 day. Liposome-encapsulated clodronate given 4 days before systemic lipopolysaccharide significantly attenuated the increases in quinolinic acid levels in blood (by 80%), liver (87%), spleen (80%), and striatum (68%) but had no effect on the increases in quinolinic acid levels in lung. These results are consistent with the hypothesis that macrophages are an important local source of quinolinic acid in brain and systemic tissues during immune activation.

The ability of clodronate-containing liposomes to deplete lymph nodes of macrophages was used as a tool to investigate the fate of liposomes in regional lymph nodes after subcutaneous (s.c.) administration. Reduced lymph node localization of liposomes in macrophage-depleted lymph nodes confirmed that phagocytosis by macrophages plays an important role in lymph node retention of liposomes. Depletion of macrophages had less effect on lymph node localization of small liposomes than on the lymph node localization of large liposomes. Inclusion of distearoylphosphatidylethanolamine (DSPE)-poly(ethyleneglycol) (PEG-PE) into the liposomes, which is known to oppose macrophage uptake, did not affect lymph node localization in macrophage-depleted or control lymph nodes. We conclude that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages. Morphological observations visualizing the uptake of PEG-liposomes by lymph node macrophages support this conclusion., (Copyright 1998 Elsevier Science B.V.)

Macrophages play an important role in host defense reactions, for example, by phagocytosis of particulate materials. This process also results in the rapid removal of targeting devices such as liposomes and adenovirus vectors and of non-autologous grafted cells and materials. Another aspect of macrophage function is their production and secretion of proinflammatory cytokines. Transient and organ-specific suppression of macrophage function by liposome-mediated manipulation has been shown to improve the efficacy of drug and gene targeting and to reduce the symptoms of inflammatory reactions.

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s-IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome-mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.

Clodronate (Cl2MBP; dichloromethylene-bisphosphonate)-containing liposomes are used to investigate the role of macrophages in immune and non-immune defense mechanisms by elimination of these macrophages followed by functional studies. The present studies characterize such liposomes in terms of the leakage of clodronate and the biodistribution of both the encapsulated drug and the liposomal carrier. The distribution of liposomes was studied using a radioactively labelled lipid phase marker (111In-DTPA-SA) and the distribution of the encapsulated drug was examined following injection of radioactively labelled clodronate (99mTc-Cl2MBP). Furthermore, the biodistribution of variously composed multilamellar liposomes (EPC/Chol molar ratio 7:1.3 or 7:7; EPC/Chol/PS 7:1.3:1 or 7:7:1; EPC/Chol/M 7:2:1; EPC/Chol/M-PE 7:2:1) after intravenous injection was investigated. The findings suggest that only one third of the originally entrapped clodronate was still encapsulated in the liposomes at the time of ingestion by the macrophages in the liver and spleen after intravenous injection of the clodronate containing liposomes. 3 h after injection no clodronate could be detected in the circulation.

Liposomes containing dioleoyl-N-(monomethoxypoly(ethylene glycol)succinyl)- phosphatidylethanolamine (PEG-PE), and of three characteristic sizes (d > 300 nm, d approximately 150-200 nm, and d < 70 nm), were prepared, injected into mice, and their biodistributions examined following a radioactive lipid phase marker. The large and small liposomes accumulated to elevated levels in spleen and liver, respectively. The intermediate sized liposomes were found to be the longest circulating. Furthermore, when injected into mice bearing murine MC-38 colon carcinoma tumor, an approximate 2-fold increase in the % injected dose per g tumor was observed for the long-circulating liposomes compared to liposomes without PEG-PE. The distribution of the injected liposomes within the tumor was examined by fluorescence microscopy, where the liposomes were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The liposomes were found surrounding blood vessels in the tumor, with some degree of extravasation into the tumor mass. A previous explanation for the reduced circulation time of small liposomes has been that they have an ability to pass through the fenestrated liver endothelium and thereby reach the parenchymal cells. DiI-labeled liposomes were therefore used to examine the intrahepatic distribution of the injected liposomes. Liposomes accumulated in liver were localized to Kupffer cells, regardless of liposome size. The small liposomes were not detectable in areas comprised of parenchymal cells when using this fluorescence technique. The reason for reduced long-circulating behavior for the small liposomes may be more directly related to the activity of PEG-PE. Therefore the steric barrier activity of the liposomes was examined by a serum protein binding assay and by streptavidin binding to biotinylated liposomes. The steric barrier was liposome size dependent, with the small liposomes revealing increased protein binding. This decreased steric barrier of the small liposomes may result in increased susceptibility to opsonization and thus explain their more rapid clearance from the circulation. The large liposomes accumulated in spleen were localized in the red pulp and marginal zone. Uptake of the large liposomes may occur by means of a filtration mechanism. These results establish the significance of liposome size in determining liposome circulation time and biodistribution, and are relevant for the optimal design of liposomes for drug delivery.

To study the role of macrophages in immune and non-immune defence mechanisms, a new technique to eliminate macrophages has been developed. This technique uses the capability of macrophages to ingest and digest particulate compounds. As particulate compound liposomes with entrapped clodronate are used. Macrophages will ingest these liposomes and after fusion of their endosomes with their lysosomes the bilayers are disrupted under influence of phospholipases and the clodronate is released into the cytoplasm. If the intracellular concentration of free clodronate reaches sufficiently high values, the macrophage will die. The applications of the approach and some of the results obtained up to now using this macrophage 'suicide' technique are discussed.

Several particulate antigens and liposomes were intravenously injected in mice in order to study their localization patterns in spleen and liver. Liposomes have been proposed as promising carriers for haptens and antigens. It was studied whether the phospholipid composition, cholesterol content and charge of the liposomes played a role in their distribution within the spleen. Different thymus-independent type 1 and type 2 and thymus-dependent particulate antigens as well as liposomes were labeled with the lipophilic fluorochrome Di-I. After labeling they were intravenously injected and spleens and livers were removed at different time intervals and prepared for light- and fluorescence-microscopy. We have observed that all particulate antigens and liposomes administered to the mice localized according to the same distribution pattern in the spleen. After 2 and 4 h particles were located in macrophages of the marginal zone and after 24 h white pulp macrophages had also ingested particulate antigens and liposomes. So we conclude that the distribution of the particulate antigens and liposomes in the spleen is independent of the immunological nature of the particles. Results are discussed with respect to the question whether or not the distribution of particulate antigens and liposome associated antigens or haptens, may be a crucial factor in determining the type of immune response to be elicited.

Dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, when injected intravenously, selectively eliminates all phagocytizing macrophages that are in direct contact with the blood circulation of the spleen and the liver. We examined whether Cl2MDP containing liposomes affect, in addition, rat monocytes and bone marrow macrophage precursors (BMMP's). In addition to Phosphatidylcholine-Cholesterol liposomes (PC liposomes), we also used mannosylated PC liposomes (PCMAN liposomes), which are reported to bind more efficiently to macrophages. Monocytes appeared to be affected 24 h after injections of 2 ml of Cl2MDP containing PC as well as PCMAN liposomes. In addition, almost no macrophages could be detected in one week cultures of blood leukocytes isolated from these animals; cultures from control animals contained +/- 50% macrophages. Bone marrow macrophage precursors did not appear to be affected.

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes. No effect of liposome-entrapped Cl2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo. This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine. These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.

The primary antibody response to intravenously administered and liposome-associated human serum albumin (HSA) was studied in mice under conditions where no response could be detected against the non-liposome-associated form of the antigen. The positive response against the antigen, entrapped in and/or exposed on the surfaces of liposomes, thus resulted from the adjuvant action of the liposomes. In mice intravenously injected with dichloromethylene diphosphonate (C12MDP) also entrapped in liposomes, all red pulp macrophages, marginal metallophilic macrophages and marginal zone macrophages had disappeared from the spleen 2 days after administration. Twenty-two days after such a treatment red pulp macrophages and marginal metallophilic macrophages had reappeared, but marginal zone macrophages were still absent. In mice injected with liposome-associated HSA at 2 days after treatment with the C12MDP liposomes, anti-HSA responses were severely depressed, but administration of the liposome-associated antigen 22 days after C12MDP liposomes elicited a normal response. These results point to a role of splenic macrophages in the processing of liposome-associated antigens, but marginal zone macrophages, which are located close to the open ends of the white pulp capillaries and thus are the first macrophages to meet the antigens arriving in the marginal zone are not required.

In the present study the tissue distribution of [3H]methotrexate was studied after intravenous injection of [3H]methotrexate-containing liposomes in normal and macrophage-depleted mice. Elimination of macrophages was performed by treatment with dichloromethylene diphosphonate- (DMDP)-containing liposomes. After thorough elimination of the macrophages from spleen and liver, by two intravenous injections of DMDP liposomes 6 and 4 days before tissue distribution studies, we found dramatic changes in the localization pattern of [3H]methotrexate liposomes in the blood, due to a decreased uptake of [3H]methotrexate liposomes by the DMDP liposome-treated liver. Because of the absence of these macrophages that are able to clear the blood of liposomes, and because of the resulting higher blood level of liposomes, we found an enhanced uptake of [3H]methotrexate liposomes by the spleen. It may be concluded that, in the spleen, apart from uptake of liposomes by macrophages, at least one other mechanism is responsible for the clearance of liposomes from the circulation. When comparing cholesterol-rich with cholesterol-poor liposomes, we found basically the same results, although uptake of cholesterol-rich liposomes by macrophages was smaller than that of cholesterol-poor liposomes, as found in several other studies. We suggest that pretreatment with DMDP liposomes can help to maintain a high level of intravenous-injected liposome-entrapped material in the blood, which otherwise would be removed by macrophages.

In the present experiments, the secondary immune response against antigens is studied after priming with liposome associated antigens and booster injections with the antigen alone, in order to study the effect of liposomes on the generation of immunological memory against the associated antigens. Liposomes show adjuvant activity with respect to both the primary and secondary immune response against associated human serum albumin (HSA). When the injected dose of liposome associated HSA was too low to elicit a primary immune response, generation of immunological memory against the antigen could not be detected. Horse radish peroxidase (HRP) associated with liposomes did not elicit a primary immune response, but immunological memory against the antigen was established.

It is shown ultrastructurally that intravenously injected and liposome-entrapped dichloromethylene diphosphonate (DMDP) causes marked damage to marginal zone macrophages in the mouse spleen which finally results in the elimination of these cells. Marginal zone lymphocytes are also affected by this treatment, probably as a result of action of the enzymes released by dying macrophages. Marginal zone macrophages largely disappear, 24 h after i.v. injection, leaving an open-meshed reticulum in which a few viable and necrotic lymphocytes are present. The proposed method to eliminate macrophages for some time may be used to study functional aspects of these cells in vivo.

Macrophages in the murine spleen were eliminated by the intravenous administration of dichloromethylene-diphosphonate (DMDP) encapsulated in liposomes. The immune response against an antigen (bovine serum albumin, BSA) or hapten (dinitrophenyl, DNP) associated with the surface of the DMDP liposomes was studied. Significant effects of macrophage elimination on the primary (IgM), but not on the secondary (IgG), anti-BSA response were found. Dramatic effects on the response against liposome-associated DNP were observed in macrophage-depleted mice. The number of anti-DNP antibody-forming cells in the spleen decreased from 300 to 28 per section, and anti-DNP serum titres dropped to 12% of their normal values. Since a similar phenomenon was observed for TNP-Ficoll, a thymus-independent type-2 antigen (and not for thymus-dependent or thymus-independent type-1 antigens), we suggest that this response should be classified as thymus-independent type-2 on grounds of its in vivo behaviour. We conclude that adjuvant activity (and memory formation) of liposomes with antigen exposed on their surface occurs irrespective of the presence or absence of splenic macrophages, and that DMDP liposomes could be useful in drug targeting with antigenic liposomes.

The immune response against HSA (human serum albumin) was studied in rabbits after intravenous injection of various HSA preparations. When HSA was injected one day after, together with or coupled to lysolecithin, a late response was found in twelve out of thirteen rabbits, whereas a minority of the rabbits responded when lysolecithin was omitted. These results confirm the adjuvant activity of lysolecithin. A rapid response starting on day 6 was found in rabbits injected with HSA entrapped in liposomes which had been composed of lecithin, phosphatidic acid and cholesterol (PPC liposomes). The response against liposome entrapped HSA was delayed for about one day when the phospholipid adjuvant lysolecithin was incorporated in the liposomes (LPPC liposomes). Results lend support to the hypothesis that the adjuvant activity of lysolecithin and its opposite inhibition of the adjuvant activity of liposomes are mediated by the same mechanism, i.e. inhibition of enzymatic digestion in lysosomes of macrophages.

Endotoxin has been shown to enhance the adjuvant effect of liposomes to protein antigens. When a weak antigen and endotoxin were associated with the same liposomes, antibody production could be detected 3 days (72 hours) after injection of the antigen-liposome-endotoxin preparation. When antigen and endotoxin were associated with different liposomes and these antigen-liposome and endotoxin-liposome preparations were injected simultaneously, the adjuvant effect of the liposomes was enhanced also, but not enough to detect antibody production on day 3.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.

The immune response to HSA-phosphatidylcholine complexes administered to rabbits was not markedly enhanced when compared with the response to unmodified HSA. It was found in earlier work that HSA entrapped in liposomes (mainly composed of phosphatidylcholine) evoked a strong immune response under conditions where no response was detected to free HSA. The present results exclude the possibility that HSA-phosphatidylcholine complexes which may arise from liposome-encapsulated HSA may be responsible for the adjuvant activity of the liposome. The adjuvant activity of liposomes could also be established after administration of a liposome-associated strong antigen (BGG).

Human serum albumin has been injected intravenously in rabbits either free in solution or associated with liposomes. Blood samples were obtained from the rabbits at various time intervals after injection, and two different antibody determinations were performed in each sample. Whereas a haemagglutination technique was applied for determination of predominantly IgM anti-human serum albumin antibodies, a second technique, using antigen-coated Sepharose beads and horseradish peroxidase-conjugated anti-rabbit IgG, was used to detect the IgG anti-human serum albumin antibodies. Liposomes appeared to enhance strongly the IgM response against human serum albumin. No such marked differences were found, however, between the IgG responses against liposome-associated or free human serum albumin. The conclusion is drawn that the immunoadjuvant effect of liposomes during the primary immune response against an albumin antigen is mainly due to an enhanced IgM antibody production.

Three ways of antigen administration to rabbits were compared with respect to the resulting immune responses. Antigen was administered free in solution, entrapped in liposomes or free in solution but together with empty liposomes. Liposomal material showed adjuvant activity when injected together with free antigen, but a much stronger adjuvant effect was found when antigen was injected entrapped in liposomes.

Dichloromethylene diphosphonate (DMDP), encapsulated in liposomes and administered intravenously in mice, will eliminate all macrophages in spleen and liver. DMDP can be incorporated in liposomes to a maximum of 15 mM, if less than 12 mM is encapsulated not all macrophages will be eliminated (since the animals could not be injected with more liposomes). To determine DMDP content of the liposomes before in vivo administration, an in vitro test system was developed. This method is based on the competition for calcium binding by either DMDP or murexide. Murexide is a metallochromatic indicator which gives a distinct wavelength shift after binding of calcium. The decrease in absorbance at 510 nm of the murexide-calcium complex, due to the addition of DMDP, was used as a reliable (s.d. 5 per cent) value for measurement of DMDP concentrations. The possible use of this system in the study of calcium binding and transport over artificial biomembranes is discussed.

Multilamellar phosphatidylcholine liposomes were coated with the antigens human serum albumin (HSA) or bovine gamma globulin (BGG) simply by suspending the liposomes in a solution of the antigens. Antigen coated phosphatidylcholine liposomes showed nearly the same adjuvant activity after intravenous injection as liposomes of a more complex phospholipid composition. Since phosphatidylcholine liposomes are biodegradable, harmless, easily obtainable, have no immunogenic activity of their own and may be administered intravenously, they seem to be a promising immunoadjuvant.

Five different ways of association of proteins with the surfaces of liposomes were compared. L-asparaginase (L-asp.) and horse radish peroxidase (HRP) were used as proteins since these show a large difference in their numbers of amino groups. Liposomal association was performed by way of: 1) non-specific coating of empty liposomes; 2) incorporation after 'hydrophobisation' with palmitoylchloride; 3) as 2 with dodecanoic acid; 4) coupling to phosphatidylinositol (PI) already incorporated in the liposomal membrane; and 5) as 4 with phosphatidylethanolamine (PE). The relative effectiveness of the different procedures for coupling of the proteins to liposomes as determined by the enzyme activities of intact liposomes shows a large variation, dependent on both the method used and the protein.

Macrophages form an important cell population involved in numerous immunological processes. Techniques which eliminate these cells, in order to permit 'in vivo' studies of their function are generally imperfect and for that reason we have developed a method using the liposome encapsulated drug dichloromethylene diphosphonate (Cl2MDP). The liposomes are ingested by the macrophages which are then destroyed following phospholipase-mediated disruption of the liposomal bilayers and release of the Cl2MDP. In the present review, technical details of the preparation of liposome encapsulated Cl2MDP are given, and applications of the technique are discussed.

Mice were injected with sphingomyelin/cholesterol or phosphatidylcholine/cholesterol (PC/C) liposomes, from twice up to 10 times, on alternate days. Administration of sphingomyelin/cholesterol (SM/C) liposomes gave rise to hepato and splenomegaly, microgranulomatous infections and changes in macrophage numbers and activity in spleen and liver. Enzyme and immuno-cytochemical methods were used, to demonstrate the effect of liposomes on the lymphoid and non-lymphoid cell populations, on cryostat sections of the spleen. Routine histological staining, of sphingomyelin/cholesterol treated animals, showed no drastic changes in morphology or compartmentalization of the spleen, apart from a small enlargement (with some microgranulomas) of the red pulp. No significant differences were found in the presence or localization of T-helper, T-cytotoxic/suppressor, T-total-lymphocytes, B-total-lymphocytes, red pulp macrophages, marginal metallophils, or non-lymphoid dendritic cells. However, a transient suppression of cells expressing marginal zone macrophage surface marker ERTR-9, was observed between the second and eighth (intravenous) administration of sphingomyelin/cholesterol liposomes. Immunization of these animals with trinitrophenyl (TNP)-ficoll, a thymus-independent type-2 antigen which is specifically processed by marginal zone macrophages (MZM), showed that these cells were not suppressed with regard to their immunological function. We conclude that chronic administration of sphingomyelin liposomes influences macrophages, probably through a general phagocytic-system overload, but not permanent or damaging changes in splenic cell populations or immunological functions occur.

It is shown in the present study that RAW 264 tumor cells can be killed by liposome-entrapped dichloromethylene diphosphonate (DMDP), both in vitro and in vivo. DMDP is ingested by phagocytic cells when entrapped in liposomes. Once phagocytized the liposomal membranes are digested and the drug is released into the cell and is ready for action. In vitro, even low doses of liposome-entrapped DMDP caused an significant reduction in cell numbers. In vivo, liposome-encapsulated DMDP markedly reduced tumor formation in the liver, when given 1 day after injection of 1 x 10(6) RAW 264 tumor cells. Liposome-encapsulated DMDP, given 4 or more days after injection of the tumor cells had no significant effect. We concluded that tumor formation by RAW 264 cells is only susceptible to in vivo treatment with liposome-entrapped DMDP during a short period of time after injection of the cells. Obviously, phagocytosis of the tumor cells is reduced after this period making the cells less susceptible to treatment with the liposome-entrapped drug.

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome-entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome-entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.

Attempts were made to study the localization of intravenously injected liposomes and enclosed labelled antigens in the spleen of mice, using autoradiography. A more than hundred fold increased uptake of antigen by the spleen was obtained when liposome entrapped antigen was compared with free antigen or antigen-antibody complexes which have been used in earlier studies. No marked accumulation of the labelled antigen was found in the follicles of the spleen, although specific antibodies to the injected antigen have been injected simultaneously with the liposome entrapped antigen. It is argued that the bulk of the labelled antigen in the spleen 0.5 to 2 h after injection was still enclosed in the liposomes, whereas part of the label, arranged in patches in the red pulp and marginal zone, corresponds with labelled antigen in macrophages, released after digestion of the liposomal membranes. The present techniques did not enable the detection of the liposomes themselves.