Trafficking of surface-linked and encapsulated liposomal antigens in macrophages: an immunocytochemical study.

Liposomal antigens are potent adjuvants of humoral and cell-mediated immunity. Although this property requires as an essential condition a physical association between the antigen and the phospholipid vehicle, the nature of the association, i.e., encapsulation or surface linkage, markedly influences the outcome of the elicited response. Available evidence suggests that macrophages are involved in this fine tuning of the immune response in a manner that is not yet clearly established. It is postulated that this might be related to their capacity to interact differently with surface-linked and encapsulated formulations. Using conalbumin as a model antigen, we address the question by analyzing the movements of encapsulated and surface-linked antigen as well as those of MHC-II molecules in macrophages in a pulse-chase immunoelectron microscopic study carried out over a 24-hr period. The antigen was followed using a polyclonal serum specifically raised against fragmented conalbumin (fCA) that allows the detection of processed antigen and of some MHC-peptide complexes. The results indicate that, in macrophages, the two liposomal formulations affect macrophage morphology in distinct ways and circulate through the various subcellular compartments with different kinetics. On the basis of the overall results, we conclude that surface-linked antigen gains access less readily to the endogenous presentation pathway than encapsulated antigen but can favor a more sustained activation of the immune system through its production of exosome-like structures and its more thorough utilization of the MHC-II pathway.

Differential activation of cell-mediated immune functions by encapsulated and surface-linked liposomal antigens.

Liposomes act as powerful adjuvants if physically associated with a protein antigen. Their effect on the immune response, however, varies with the nature of this linkage, surface-linked and encapsulated antigens having different properties. Cytometric analysis and cytokine measurements indicate that this difference may be due to the differential activation of T lymphocyte populations. Surface-linked antigen appears to preferentially stimulate CD4+ T cells to proliferate and mature into a typical Th1 phenotype; this is indicated by a positive shift in the CD4+/CD8+ ratio of sensitized splenocytes, a massive production of interferon-gamma, and the absence of interleukin-4 secretion. In contrast, encapsulated antigen, while stimulating spleen cell proliferation, does not significantly affect the CD4+/CD8+ ratio and induces only low levels of interferon-gamma production in the absence of interleukin-4 secretion. These results suggest that CD4+ and CD8+ populations are both expanded in response to encapsulated antigen but that neither typical Th1 nor Th2 phenotypes are induced. High-resolution immunocytochemical investigations show that this differential activation of T cell populations may be related to a different intracellular trafficking of antigens into professional antigen-presenting cells. Whereas surface-linked antigen remains predominantly in endosomal compartments where it may be associated with major histocompatibility (MHC) class II products for presentation to CD4+ T cells, encapsulated antigen escapes into the cytosol, reaching the MHC class I pathway for presentation to CD8+ T cells. The results therefore suggest that both liposomal antigens stimulate cell-mediated immunity albeit differently. This behavioral difference may be of practical importance in the design of adjuvants for the preferential potentiation of specific cytotoxic effector functions.

Differential biodistribution of encapsulated and surface-linked liposomal antigens.

The biodistribution of liposomal antigens either encapsulated in or surface-linked to liposomes of similar composition was studied over time following intravenous injection and the results analyzed in relation to adjuvanticity. The two formulations were shown to behave very differently in vivo. While encapsulated antigen was rapidly focused to liver and spleen as expected, surface-linked antigen exhibited a more disseminated distribution which parallels that of the free protein. In dual-labelling experiments, it was also shown that encapsulated antigen remains associated with its liposomal vehicle in contrast to surface-linked antigen which is rapidly dissociated. This dissociation was apparently neither due to an exchange with plasma lipoproteins nor to a direct action of blood constituents. Besides, it was found that surface-linked antigen was rapidly accumulated in the carcass. We propose that the retention of the surface-linked antigen in the carcass results from a pre-processing of the protein involving more probably mononuclear phagocytes. This pre-processing might in turn favor the dissociation of the protein from the liposomes in a form that allows its dissemination in the whole organism and its interaction with more efficient antigen presenting cells such as for example Langerhans or dendritic cells.

Cytometric profile of molybdenum-induced contact sensitization versus a strong allergen reaction to oxazolone in murine auricular lymph node (ALN) test.

Induction of contact hypersensitivity (CH) by molybdenum chloride (MoCl5) was determined by auricular lymph node (ALN) test in C57B1/6 mice. The ALN test was further improved by immunophenotyping and cytometric analysis of subset-specific cell in the draining node. Skin sensitization was induced by topical ear exposure to 1.0-50% oxazolone and resulted in a strong dose-related ALN reaction. Analogous exposure to MoCl5 resulted in a weaker but marked dose-related reaction, also manifested as an increase in cell number/ALN. Other differences between the oxazolone-induced strong sensitization and the MoCl5-related ALN reaction were: (1) an increase in the total number of Ig+ cells, which was, however, unchanged in the MoCl5-exposed mice; (2) a significant increase in the total number of large/activated T-cell subsets; and (3) a marked shift in the relative percentage of gated large/activated subsets of ALN cells, which was not observed in the MoCl5-exposed animals. Thus, it appeared that the molybdenum exposure induced a nonspecific increase in the cell number/ALN and was not accompanied by any marked activation of the T-cell subsets. Immunotoxicity of a 14 day subchronic exposure to MoCl5 at 1-100 ppm in food was studied by quantification of splenic humoral IgM response to sheep erythrocytes (SRBC). Plaque-forming cells (PFC) and enzyme-linked immunosorbent assay (ELISA) revealed unchanged humoral exposure in MoCl5-exposed mice. Cytometric assay of fluorescent beads uptake showed unchanged phagocytic activity of peritoneal macrophages from the MoCl5-exposed mice. Immunophenotyping of CD4+, CD8+, Thy 1.2+ and Ig+ cells revealed no effect of MoCl5 exposure on the total count of cell subsets in the ungated populations of spleen, lymph nodes and peripheral blood cells. Molybdenum chloride should thus be considered as a non-immunotoxic and a weak, nonspecific contact irritant.

Immunoactivating potential of multilamellar liposome vesicles (MLV) in murine popliteal lymph node (PLN) test.

Immunoactivating properties of subcutaneously injected small unilamellar vesicles (SUV) and multilamellar liposome vesicles (MLV) were studied in relation to different transition temperatures (Tc) of phospholipids. Liposome-induced proliferative reaction in the popliteal lymph node (PLN) was quantified by subsequent cytometric assay. Early cell activation during the onset of PLN reaction was monitored by immunophenotyping of lymphocyte subsets stained with a panel of monoclonal antibodies (mAbs) and gating the subset-specific large/activated cells. Injection of MLV liposomes containing distearoyl phosphatidylcholine (DSPC) and dipalmityl phosphatidylcholine (DPPC), characterized by relatively high Tc, resulted in a marked PLN reaction, increased numbers of CD4+, CD8+, Ig+ subsets and increased proportions of large/activated EAM+ (CD69+) and CD25+ (IL-2 receptor+) cells. The reaction was dose and time dependent. In contrast, injection of MLV liposomes containing lipids of low Tc, such as egg phosphatidylcholine (egg PC) and dimyristoyl phosphatidylcholine (DMPC), did not show any immunoactivation. In addition, there was a highly reduced immunoactivating potential of small-size SUV liposomes over large-sized MLV of identical phospholipid composition. Generally, both lipid composition and vesicle size appeared to be essential for the immunoactivating potential of liposomes. The data suggest a possible correlation between the Tc of the phospholipid and the immunoactivating potential of the large-sized MLV liposomes.

Liposomal adjuvanticity: effect of encapsulation and surface-linkage on antibody production and proliferative response.

The immunoadjuvanticity of liposomal antigens, namely encapsulated and surface-linked conalbumin, was studied at different levels of an immune response including immunoglobulin production, blastogenic response and lymphokine production in sensitization conditions compatible with vaccine designs. The results demonstrated that both liposomal formulations stimulate all properties analyzed with respect to free antigen but significantly differ in some of their inductive capabilities, suggesting that they follow different routes in the immune network. Thus, although both liposomal antigens are capable of inducing potent humoral responses characterized by increased production of IgM and IgG2a, covalently linked antigen stimulates a quasi-polyclonal blastogenic response accompanied by the simultaneous secretion of IL-2 and IFN gamma, while encapsulated antigen which is less blastogenic mainly induces IL-2 secretion. We can conclude that: first, both antigenic formulations induce a Th1 type of activation and might therefore potentiate cell-mediated immunity, but surface-linkage favors a more rapid maturation of the response and a much more intense help induction. Second, although a strong adjuvanticity can be observed whatever the route of sensitization, namely intraperitoneally, intravenously or subcutaneously, the intravenous injections induce the better potentiation. The in vitro data were all compared to those obtained with naïve mice, allowing dissociation of the contribution of the in vitro rechallenge from that of the in vivo immunization. These differences observed between the liposomal antigens might be taken advantage of while formulating vaccines specifically suited to meet required needs and suggest that covalently linked antigen might be particularly useful in situations where induction of cell-mediated immunity is of prime importance.

Lymphocyte activation by liposome-trapped streptozotocin in murine popliteal lymph node (PLN) test.

The lymphoproliferative potential of liposome-trapped streptoztocin (STZ) was compared to the effect of saline-dissolved STZ injected locally into the foot pad of CD-1 mice. Popliteal lymph node (PLN) enlargement and early cell activation of lymphocyte subsets were monitored during the onset of STZ-induced autoimmune-like reaction. Injection of the optimal STZ dose, 0.5 mg/foot pad, markedly increased the absolute PLN cell number as well as specific T-helper (CD4+), T-suppressor/cytotoxic (CD8+), and B-(Ig+) cell subsets stained with fluorescent monoclonal antibodies. Furthermore, there was a marked increase in the number of large/activated CD4+ and CD8+ cells and subsets bearing specific markers of early activation. These included cells stained with fluorescein-conjugated monoclonal antibodies against interleukin-2 receptor (CD25+) and early activation marker (EAM+) (CD69+), and with fluorescein-conjugated peanut agglutinin (PNA+). Surprisingly, the injection of liposome-trapped STZ, at a 1/10 of the optimal dose only, induced a marked PLN enlargement comparable to the effect of optimal STZ dose. The effect of liposome-STZ could be dissociated from the non-drug-containing MLV-related lymphocyte activation. The data suggest several possible advantages from the introduction of chemicals by the liposome route and the subsequent PLN test for chemical-induced autoimmunity. Toxicological advantages could involve better control of chemical exposure, controlled exposure to the water-insoluble substances, drastic reduction of xenobiotic dose, a stronger, clear PLN response and possible elimination or at least restriction of false-negative results, due to the liposome adjuvancity. Overall, application of liposomes as an exposure route potentialized the STZ-induced early lymphocyte activation.

Correlation between in vitro and in vivo behaviour of liposomal antigens.

Using conalbumin as a model antigen, we demonstrate in this paper that liposomal antigen differently influences the activation of the immune system depending on the mode of association of the antigen with the liposomal vehicle whether it is by encapsulation or surface linkage. This conclusion is based on in vivo data showing that encapsulated antigen induces a short-lasting response dominated by IgG1 production while surface-linked antigen has a longer-lasting effect characterized by increased production of IgM, IgG2a, IgG3 as well as of IgG1. The in vivo data were complemented by in vitro proliferation studies carried out on spleen cells or macrophage-depleted spleen cells obtained from mice sensitized in vivo and rechallenged in vitro on day 4 following sensitization. Rechallenge was carried out in the absence or presence of anti-IL1. The data indicate that, in contrast to what is generally observed in vivo, liposomes alone potentiate spleen cell proliferative response in a dose-dependent manner. This liposomal effect totally obscures the antigen-specific proliferation that was expected with encapsulated antigen without masking that induced by surface-linked antigen. The mode of antigen association also influences anti-cytokine responsiveness as demonstrated by the insensitivity of the surface-linked antigen response to the presence of anti-IL1 and the significantly decreased response observed with encapsulated antigen under identical conditions. The response to both liposomal antigenic formulations was almost totally abolished in adherent cell-depleted cultures. The overall results therefore suggest that encapsulated and surface-linked antigens activated different immune pathways.

Modulation of amphotericin B activity by association with mannose ester.

The biological and molecular properties of a new formulation of Amphotericin B complexed with the surfactant palmitoyl mannose were studied in in vitro as well as in in vivo situations. The properties analyzed include toxicity towards two types of mammalian cells and four fungi strains, effect on macrophage activity, inflammatory properties, acute toxicity in mice and spectral behavior in presence of foetal calf serum or 6% propanol. The results demonstrate that, in presence of palmitoyl mannose, the cytotoxicity of AmB is decreased towards both, fungal and mammalian cells while its fungistatic potential is increased, its inflammatory properties are conserved and its acute toxicity is significantly diminished. These effects can be potentially explained by the formation of a complex between AmB and the sugar ester that impedes the interaction of the drug with either serum components or cell membrane constituents. The overall properties of AmB in the complex would be expected to favor an increase in the immunoadjuvant properties of the drug, a more localized inflammation during fungal infection and consequently a better therapeutic efficiency.

Liposomal dexamethasone effectiveness in the treatment of hypersensitivity pneumonitis in mice.

The effects of daily intranasal instillation of liposomal dexamethasone and free dexamethasone phosphate were compared in a murine model of hypersensitivity pneumonitis induced by Saccharopolyspora rectivirgula (formally known as Micropolyspora faeni). After 3 weeks of antigen and liposome instillations, lung response was evaluated by bronchoalveolar lavage cell counts, lung index and histopathology. Systemic absorption was evaluated by measuring plasma adrenocorticotropic hormone (ACTH) level. Free dexamethasone phosphate induced a dose-dependent response with the maximal effect reached at 1 mg kg-1. At 0.1 mg kg-1, liposomal dexamethasone had a greater effect than free dexamethasone phosphate on bronchoalveolar cells ml-1: 3.01 x 10(5) +/- 0.35 x 10(5) compared to 4.70 x 10(5) +/- 0.34 x 10(5), and lung index: 1.22 +/- 0.10 compared to 1.86 +/- 0.07. Effect on histopathology was similar. Plasma ACTH levels (pg ml-1) were: 75.1 +/- 14.0 for animals receiving antigen and free dexamethasone phosphate (0.2 mg kg-1), and 149.7 +/- 12.0 for animals receiving antigen and liposomal dexamethasone (0.2 mg kg-1). In conclusion, liposome-incorporated dexamethasone is efficient in the treatment of experimental hypersensitivity pneumonitis and, contrarily to free dexamethasone phosphate, does not inhibit ACTH secretion.

Mechanism of liposome adjuvanticity: an in vivo approach.

The reputation of liposomes as adjuvant of the immune response is now firmly established despite the lack of information on the mechanisms involved in their immunopotentiating properties. The rapid targeting of massive doses of antigenic material to antigen-presenting cells, especially macrophages has, however, often been invoked as the principal source of liposomal adjuvanticity. In order to test this hypothesis, we analyzed the humoral response to antigen encapsulated in liposomes containing increasing amounts of surface-exposed mannose residues, ligand specific of an exclusive macrophagic receptor. Using BSA as a model antigen, we demonstrated that the humoral response is profoundly affected by mannosylation, being of prolonged duration and either inhibited or activated depending on the immunizing doses. These results suggest that the rapidity of antigen targeting is not the sole reason to liposome adjuvanticity and that the role of liposomes as antigenic depot is probably important to sustain substantial activation through successive restimulations. In this context, the increased rapidity in antigen targeting which favors the concentration of activation signals in time, results in an under-optimization of the response at high immunizing doses and in an optimization of this response at doses that would otherwise give rise to signal of sub-threshold intensity albeit during a longer period of time.

Liposome adjuvanticity: influence of dose and protein:lipid ratio on the humoral response to encapsulated and surface-linked antigen.

The humoral response to bovine serum albumin either encapsulated in or surface-linked to liposomes was studied as a function of dose and protein:lipid ratio. Total immunoglobulin, total IgG, IgM, and the G isotypes, IgG1, IgG2a, and IgG3 were measured during the plateau phase of production after a boosting injection. Although the adjuvant character of liposomes was confirmed regardless of the mode of antigen association, important differences in the response to the two types of liposomal formulations were observed. Our results suggest that surface-linked antigen stimulates the immune system at lower doses than its encapsulated counterpart, is more sensitive to the protein:lipid ratios, and can stimulate the production of particular immunoglobulin isotypes in controlled conditions. Our data support the idea that different pathways of processing are utilized by the two forms of liposomal antigen.

Liposomal vaccine: influence of antigen association on the kinetics of the humoral response.

The kinetics of the primary and secondary humoral responses to either encapsulated or surface-linked bovine serum albumin have been compared by measuring the production of specific total immunoglobulin, IgG and IgM at various times postimmunization. From our data it can be concluded that surface linkage is the best way to induce a rapid, intense and prolonged response which, in contrast to that induced by encapsulated BSA, is characterized by a low IgG/IgM ratio. The results are discussed in relation to the possible routes followed by the antigen depending on its mode of association with liposomes in the initiation of the humoral response. Our results suggest that liposomal vaccine may be designed to activate specific pathways of the immune network preferentially.

Modulation of CD4 expression on lymphoma cells transplanted to mice fed (n - 3) polyunsaturated fatty acids.

Groups of adult AKR mice were fed well defined fats controlled diet regimens. These consisted of either saturated (beef tallow: 'BT') or (n - 3) polyunsaturated (fish oil: 'FO') fatty acids supplementation to basal mix mouse food. In other groups, the basal mix was given without any fat supplement ('NF'). Six weeks or more after the initiation of these diet regimens, mice received intraperitoneal injection of histocompatible RDM-4 lymphoma cells. Ascites RDM-4 tumors were harvested approximately two weeks later, and some of their physicochemical properties were studied. It was repeatedly found that: (1) the tumor grew considerably faster in the FO-fed donor than in the BT- or NF-fed donors; (2) cell membrane fluidity, content of C20(n - 3) and of C22(n - 3) fatty acids were significantly higher in the FO groups than in both BT and NF groups, while the content of C20(n - 6) and 22:4(n - 6) fatty acids was concomitantly decreased; (3) expression of the CD4 cell surface marker was always significantly diminished in the FO groups, whereas other markers such as CD8, H2K, Thy-1 and LFA-1 were not affected. Similar results were obtained, whether fats constituted from 1% to 16% by weight of the food intake. Use of a recently selected line of the RDM-4 lymphoma, exhibiting higher CD4 marker expression, resulted in similar observations. On the other hand, CD4 expression on cells from lymphoid organs of healthy adult AKR mice was not detectably modulated by the dietary fats.

Pleiotropic effects of positively charged liposomes on immune functions.

Positively charged liposomes have been shown to inhibit the proliferation of lymphocytes induced by various polyclonal activators. We demonstrated that this inhibition is essentially restricted to early phases of activation. B cell proliferation, induction of suppressor cells, and cytotoxic activities are all profoundly inhibited, whereas T4+ cells response to mitogenic stimulation is only moderately affected. The results are discussed in terms of membrane perturbations potentially induced by liposome-lymphocyte interactions.

Importance of physical association between antigen and liposomes in liposomes adjuvanticity.

The importance of a physical association between antigen and liposomes in liposomes adjuvanticity has been evaluated by comparing the anti-BSA secondary humoral response induced by different BSA formulations including free antigen, free antigen + liposomes, or encapsulated antigen. Our results demonstrate that a physical association of antigen with liposomes is required for a potentiation of the humoral response to be observed and that empty liposomes, when used for priming together with free antigen, restrain the induced response, as shown by a slower triggering, a reduced intensity and a more rapid decay rate. Free BSA has an intermediate behavior and, in contrast to what is observed with BSA/liposomes preparations, its effect appears to be dose-dependent. Each of the three BSA formulations also differs in the relative production of IgG and IgM they induced during the response.

Comparative effect on humoral response of four different proteins covalently linked on BSA-containing liposomes.

The humoral response to encapsulated BSA appears to be a classical TD antigen response with a high ratio of IgG to IgM, whereas that to covalently-linked antigen is more complex, characterized by an enhanced synthesis of IgM, leading to an equal production of IgM and IgG. In a recent paper, we observed that surface-linked Con A on BSA-containing liposomes changed the isotype distribution to encapsulated BSA so as to mimic the response to surface-linked antigen. In the present study, we compared the immune response to BSA in BALB/c mice immunized with the antigen encapsulated into liposomes coated with one of four different proteins: Con A, Myo, MSA, or PWM. The humoral response was analyzed by measurements of antibody production (total Ig, IgM, and IgG isotypes) on serum samples obtained by cardiac puncture. It can be concluded from our results that any surface-linked protein may affect the interaction between liposome-associated antigen and immunocompetent cells.

Stimulation of lymphoproliferation by sucrose esters.

Lauryl sucrose has recently been shown to decrease the toxicity of amphotericin B (AmB), a widely used antifungal agent, probably through a modulation of its physical state. In this paper, we demonstrate using a lymphoproliferative assay, that lauryl sucrose and monosubstituted analogs in addition to their effects on AmB also possess significant immune enhancer properties. An anti-bell shape relationship was observed between stimulation of lymphoproliferation and chain length of ester's fatty acid, lauryl sucrose being the less active of the tested derivatives. These properties of sucrose esters are discussed in the specific context of antifungal therapy.

Immunopotentiation of the humoral response by liposomes: effect of a T cell polyclonal activator.

In this paper, we analyzed the influence of surface-linked Con A on the secondary response to liposome-associated antigen via either encapsulation or covalent linkage at the liposomal surface. The study was carried out on BALB/c mice using bovine serum albumin as antigen. The humoral response was evaluated by measurements of antibody-producing cells (total, IgM, and IgG) and serum antibody titers. The results indicate that Con A at submitogenic concentrations does not potentiate the overall effect of liposomes but drastically changes the isotype distribution pattern obtained in response to encapsulated antigen without however affecting that obtained in response to surface-linked antigen. In all situations where Con A and/or BSA was covalently linked, IgG and IgM isotypes were produced in equal quantity, while in response to encapsulated BSA, IgG was by far the dominant isotype produced as expected for a thymo-dependent antigen. These results suggest that the quality of an immune response and the mechanisms of activation may be profoundly influenced by the nature of antigen association with liposomes as well as by the presence at the liposomal surface of immunomodulators such as Con A.

Immunopotentiation of the humoral response by liposomes: encapsulation versus covalent linkage.

Two different modes of antigen association with liposomes were compared for their stimulation of IgM- and IgG-producing cells in primary- and secondary-response experiments. The study was carried out on BALB/c mice using the antigen bovine serum albumin either free, encapsulated in liposomes or covalently linked to the liposomal surface. Our results indicate that, although both types of liposome association are equally efficient in potentiating the humoral response, encapsulation mainly favours IgG isotype production with little or no effect on the IgM subset, while covalent linkage stimulates the production of both IgG and IgM. Our results reconcile some apparently conflicting published data and suggest that the mode of antigen association with liposomes considerably influences the pathways by which stimulation occurs.