744
Bioconjugate Chem. 2002, 13, 744−749
Cholesterol Inclusion in Liposomes Affects Induction of Antigen-Specific IgG and IgE Antibody Production in Mice by a Surface-Linked Liposomal Antigen Yoshio Nakano,† Masahito Mori,† Hiroyuki Yamamura,† Seishiro Naito,‡ Hiroshi Kato,‡ Maiko Taneichi,‡ Yuriko Tanaka,‡ Katsutoshi Komuro,‡ and Tetsuya Uchida*,‡ DDS Development Division, NOF Corporation, Tokyo, Japan, and Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan. Received October 15, 2001; Revised Manuscript Received April 24, 2002
In the previous study, we investigated the induction of ovalbumin (OVA)-specific antibody production in mice by OVA-liposome conjugates made using four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids. All of the OVA-liposome conjugates tested induced IgE-selective unresponsiveness. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes with the highest membrane fluidity, suggesting that the membrane fluidity of liposomes affects their adjuvant effect. In this study, liposomes with five different cholesterol inclusions, ranging from 0% to 43% of the total lipid, were made, and the induction of OVA-specific antibody production by OVA-liposome conjugates was compared among these liposome preparations. In contrast to the results in the previous study, liposomes that contained no cholesterol and possessed the lowest membrane fluidity demonstrated the highest adjuvant effect for the induction of IgG antibody production. In addition, when the liposomes with four different lipid compositions were used, OVA-liposome conjugates made using liposomes that did not contain cholesterol induced significantly higher levels of anti-OVA IgG antibody production than did those made using liposomes that contained cholesterol and, further, induced significant production of anti-OVA IgE. These results suggest that cholesterol inclusion in liposomes affects both adjuvanticity for IgG production and regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes.
INTRODUCTION
Vaccines are undoubtedly the most efficient tool for control or elimination of severe infectious diseases. Over the course of development of modern vaccines, weak immunogenicity presented a major difficulty (1). Consequently, the use of adjuvants became indispensable. Aluminum-containing compounds are, at present, the only adjuvants approved for clinical use. However, they are not active with all immunogens, and they are known to induce production of IgE antibodies, which can cause allergic reactions in the vaccinee (2). To date, many adjuvant formulations have been developed, but only a few have been evaluated in clinical trials, mainly due to their toxicity and side effects (3). Among the candidates for adjuvants, liposomes have garnered recent attention for their capacity as carriers of vaccines (4-8). Most of the liposomal vaccines proposed have been made by antigen entrapment within the liposomal compartment (5). However, encapsulated and surface-linked liposomal antigens are known to induce differential immune responses (9). The capability to induce cellular immune response by a surface-linked, but not by encapsulated, liposomal antigen has been reported (8). * To whom correspondence should be addressed: National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama City, Tokyo 208-0011, Japan. Phone: +81 425 61 9724. Fax: +81 425 61 9722. E-mail:
[email protected]. † NOF Corporation. ‡ National Institute of Infectious Diseases.
We previously reported that surface-linked liposomal antigen induced antigen-specific IgG, but not IgE, antibody production (10). This antigen modification was expected to serve as a novel protocol for the development of vaccines that would induce minimal allergic response. IgE-selective unresponsiveness was induced regardless of the coupling procedure of antigen and liposomes (11). In addition, differential adjuvant effects were obtained with antigen-liposome conjugates including differentially formulated liposomes, whereas IgE antibody production was not induced by the prepared conjugates (12). Inclusion of cholesterol in the liposome formulation is known to affect the stability and adjuvanticity of liposomes (7, 13). In this study, liposomes with different cholesterol inclusions were prepared and then coupled with antigens in order to investigate the induction of antigen-specific IgG and IgE antibody production. The aim of this study was to determine the correlation between cholesterol inclusion in liposomes and the induction of antibody production by surface-linked liposomal antigens. MATERIALS AND METHODS
Experimental Animals. BALB/c mice (8 weeks of age, female) were purchased from Charles River (Yokohama, Kanagawa, Japan). Chemicals. All phospholipids were obtained from NOF Co., Tokyo, Japan. Reagent grades of cholesterol and 1,6-diphenyl-1,3,5-hexatriene (DPH) were purchased from Wako Pure Chemical Co., Osaka, Japan.
10.1021/bc0155667 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/27/2002
Differential Adjuvanticity in Lipsomes
Bioconjugate Chem., Vol. 13, No. 4, 2002 745
Table 1. Liposomes with Different Cholesterol Inclusions cholesterol inclusion (%) 0
10
20
30
43
liposome formulation
molar ratio
dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol
8.0 6.0 2.0 0.0 7.1 5.3 2.0 1.6 6.2 4.6 2.0 3.2 5.3 3.9 2.0 4.8 4.0 3.0 2.0 7.0
Table 2. Liposomes Made Using Four Different Carrier Lipids with or without Cholesterol name oleoyl (+CHO)
oleoyl (-CHO)
myristoyl (+CHO)
myristoyl (-CHO)
palmitoyl (+CHO)
palmitoyl (-CHO)
stearoyl (+CHO)
stearoyl (-CHO)
liposome formulation
molar ratio
dioleoyl phosphatidyl choline (DOPC) dioleoyl phosphatidyl ethanolamine (DOPE) dioleoyl phosphatidyl glycerol (DOPG) cholesterol dioleoyl phosphatidyl choline (DOPC) dioleoyl phosphatidyl ethanolamine (DOPE) dioleoyl phosphatidyl glycerol (DOPG) cholesterol dimyristoyl phosphatidyl choline (DMPC) dimyristoyl phosphatidyl ethanolamine (DMPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol dimyristoyl phosphatidyl choline (DMPC) dimyristoyl phosphatidyl ethanolamine (DMPE) dimyristoyl phosphatidyl glycerol (DMPG) cholesterol dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dipalmitoyl phosphatidyl glycerol (DPPG) cholesterol dipalmitoyl phosphatidyl choline (DPPC) dipalmitoyl phosphatidyl ethanolamine (DPPE) dipalmitoyl phosphatidyl glycerol (DPPG) cholesterol distearoyl phosphatidyl choline (DSPC) distearoyl phosphatidyl ethanolamine (DSPE) distearoyl phospatidyl glycerol (DSPG) cholesterol distearoyl phosphatidyl choline (DSPC) distearoyl phosphatidyl ethanolamine (DSPE) distearoyl phospatidyl glycerol (DSPG) cholesterol
4 3 2 7 8 6 2 0 4 3 2 7 8 6 2 0 4 3 2 7 8 6 2 0 4 3 2 7 8 6 2 0
Liposomal Antigen. Ovalbumin (OVA, Grade VII) was purchased from Sigma (St. Louis, MO). Liposomes. The liposomes with different cholesterol inclusions used in this study are listed in Table 1. The liposomes consisting of four different components, with or without cholesterol, are listed in Table 2. The crude liposome solution was passed through a membrane filter (Nucleopore polycarbonate filter, Coster, Cambridge, MA) with a pore size of 0.2 µm. The diameter of liposomes was analyzed using a NICOMP 370 submicron particle sizer (Pacific Scientific, Silver Springs, MD). After filtration, the mean size and SE of liposomes listed in Tables 1 and 2 were 222.8 ( 10.1 and 224.0 ( 14.3 nm, respectively. Preparation of Fluorescence-Labeled Liposomes. Fluorescence-labeled liposomes containing phospholipids were prepared as follows: DPPC, DPPE, DMPG, and cholesterol with the indicated molar ratio (Table 1) were
dissolved in a mixture of solvents (chloroform/methanol/ water ) 65/25/4 by volume), and then 4.3 M DPH in chloroform solution was added. After vigorous mixing, solvents were removed completely under reduced pressure, followed by the addition of 1 mM phosphate buffer (pH 6.0) containing 8.5% sucrose to create a lipid vesicle suspension. The suspension was then extruded through a polycarbonate filter (0.2 µm) to adjust the liposome size. Fluorescence-labeled liposomes containing phospholipids that included a myristoyl group, palmitoyl group, or stearoyl group acting as the acyl chain were prepared by the same method. Fluorescence Measurement. Fluorescence depolarization of DPH was measured according to the method described by Lents et al. (14) using a Hitachi F-2000 spectrophotometer equipped with a temperature-controlled cuvette holder. DPH was excited at 360 nm and its fluorescence was measured at 428 nm using an UV-
746 Bioconjugate Chem., Vol. 13, No. 4, 2002
Nakano et al.
39 instrument filter UV-39 to cutoff wavelengths below 390 nm. The degree of fluorescence polarization was calculated from the formula
P ) (IVV - IVHG)/(IVV + IVHG) where IVV and IVH are the observed fluorescence intensities measured with polarizers parallel and perpendicular to the vertically polarized exciting beam, respectively, and G is a correction factor for the transmission efficiency of vertically and horizontally polarized light. A 3-mL portion of 1 mM phosphate buffer (pH 6.0) containing 8.5% sucrose was mixed well with 0.3 mL of the liposome suspension prepared as described previously. The mixture was then allowed to stand at 37 °C for 5 min before measurement. Samples from each liposome suspension were prepared in triplicate. Coupling of OVA to Liposomes. Liposomal conjugates with OVA (OVA-liposome) via glutaraldehyde were prepared as follows: to a mixture of 90 mg of liposomes and 6 mg of OVA in 2.5 mL phosphate buffer (pH 7.2), 0.5 mL of 2.5% glutaraldehyde solution was added in drops. The mixture was stirred gently for 1 h at 37 °C, and then 0.5 mL of 3 M glycine-NaOH (pH 7.2) was added to block the excess aldehyde groups. The mixture was then incubated overnight at 4 °C. The liposome-coupled OVA and uncoupled OVA in the resulting solution were separated using CL-4B column chromatography (Pharmacia Fine Chemical Co., Upsala, Sweden). The amount of lipid in the liposomal fraction was measured using a phospholipid content assay kit (Phospholipid-Test-Wako, Wako Pure Chemical Co., Osaka, Japan). The OVA-liposome solution was adjusted to 10 mg lipid/mL and kept at 4 °C until use. For the measurement of OVA coupled to liposome, radiolabeled OVA ([methyl-14C], purchased from New England Nuclear, Boston, MA) was mixed with cold OVA and then used for coupling with liposome and for determining the calibration curve. The radioactivity of the resulting OVA-liposome solution was counted using a calibration curve. The mean and SE of OVA coupled to liposomes listed in Tables 1 and 2 were 47.0 ( 2.2 and 47.8 ( 2.5 µg/mg of lipid, respectively. These results suggested that the variation in moles of phosphatidyl choline (4-8), phosphatidyl ethanolamine (3-6), and differential cholesterol inclusion in liposomes did not result in the differential coupling efficiency of OVA to liposomes. Immunization. Mice were immunized intraperitoneally (ip) with OVA-liposome at a dose of 2 mg of lipid/ 200 µL/mouse. Four weeks after primary immunization, mice were boosted in the same manner as that used for primary immunization. For the measurement of serum antibodies, mice were bled from the tail vein. Measurement of Serum Antibodies. Six weeks after primary immunization, serum anti-OVA IgG and IgG isotypes were determined by enzyme-linked immunosorbent assay (ELISA) using HRP-labeled goat anti-mouse IgG (Zymed Lab., San Francisco, CA). OVA-specific IgE was determined by monoclonal antibody-captured ELISA using monoclonal antibody against murine IgE produced by clone B1E3. Preparation of SAC and CD4+ T Cells. Spleen cell suspensions were prepared in RPMI-1640 containing 10% fetal calf serum (FCS). Cells (5 × 107) in 5 mL of medium containing 10% FCS were plated into 50-mm plastic tissue culture dishes (No. 3002, Becton-Dickinson Labware, Franklin Lakes, NJ) and incubated at 37 °C in a humidified 5% CO2 atmosphere for 2 h. After culture, nonadherent cells were removed by vigorous washing in
Figure 1. Degree of fluorescence polarization in the liposomes with five different cholesterol inclusions. The degree of fluorescence polarization at 37 °C was calculated as described in Materials and Methods. Data represent mean and SE of the triplicate measurements. (*) Significant (p < 0.01).
warm media, and adherent cells were then harvested with a cell scraper. CD4+ T cell purification from SC of mice immunized with OVA-liposome was performed with the magnetic cell sorter system MACS, according to the manufacturer’s protocol using anti-CD4 antibodycoated microbeads (Miltenyi Biotec GmbH, No. 492-01). CD4+ T cells were suspended in RPMI-1640 containing 10% FCS at a cell density of 2 × 106/mL. In the preliminary examinations, OVA at a final concentration of 1 mg/mL, 24 h (Th1) and 96 h (Th2) culture period, respectively, were found to be optimal for cytokine production. The cell suspension was plated at 250 µL per well onto a 48-well culture plate (No. 3047, BectonDickinson Labware, Franklin Lakes, NJ), and 500 µL of 2 mg/mL OVA solution and 250 µL of 8 × 105/mL SAC in the same media was added. After incubation in a CO2 incubator, the culture supernatants were collected and assayed for cytokines. Cytokine Assays. IL-4 in the culture supernatant was measured using the Biotrak mouse ELISA system (Amersham International, Buckinghamshire, U.K.). All test samples were assayed in duplicate, and the standard error in each test was always less than 5% of the mean value. Statistical Analysis. The Student’s t-test was employed for the statistical analysis. RESULTS
Membrane Fluidity of Liposomes. Figure 1 shows the degree of fluorescence polarization of the liposome preparations listed in Table 1. A significant difference was observed in the degree of fluorescence polarization among liposomes with different cholesterol inclusions. The increase in fluorescence polarization values can be interpreted as the result of a decrease in mobility of the hydrophobic region of phospholipid bilayers in the membranes. The results suggest that the membrane fluidity of liposomes correlated well with the amount of cholesterol contained, and liposomes containing no cholesterol showed the lowest membrane fluidity. Anti-OVA Antibody Production in Mice Immunized with OVA-Liposome Prepared Using Liposomes with Different Cholesterol Inclusions. Lipo-
Differential Adjuvanticity in Lipsomes
Figure 2. Anti-OVA antibody production in mice immunized with OVA-liposome made using liposomes with five different cholesterol inclusions. Six weeks after primary immunization, mice were bled from the tail vein, and serum anti-OVA IgG were measured. Data represent mean and SE of five mice per group. (*) Significant (p < 0.01).
somes in Figure 1 were coupled with OVA and inoculated into mice as described in Materials and Methods. AntiOVA IgE antibody production was not observed in any of the groups of mice throughout the observation period (data not shown). On the other hand, anti-OVA IgG antibody production was induced in correlation with cholesterol inclusion in liposomes (Figure 2), and OVAliposome prepared using liposome that contained no cholesterol induced the highest level of anti-OVA IgG antibody production. Anti-OVA Antibody Production in Mice Immunized with OVA-Liposomes Made Using Liposomes with Four Different Lipid Formulations with or without Cholesterol. Liposomes of four different lipid formulations were prepared in the presence or absence of cholesterol as listed in Table 2, and the induction of anti-OVA IgG antibody production by OVA-liposome was investigated. OVA-liposome prepared using lipo-
Bioconjugate Chem., Vol. 13, No. 4, 2002 747
some consisting of unsaturated carrier lipids (“oleoyl” liposomes) induced the highest titer of anti-OVA IgG. Among OVA-liposomes prepared using liposomes consisting of saturated carrier lipids, the preparation using liposomes possessing the longest carbon chain (“stearoyl” liposomes) induced the lowest IgG antibody production when cholesterol was included in the liposomes (Figure 3a). On the other hand, OVA-liposome prepared using liposomes that did not contain cholesterol induced a significantly higher level of anti-OVA IgG antibody production than did those prepared using liposomes that contained cholesterol, except in the case of “oleoyl” liposomes. Interestingly, significant anti-OVA IgE antibody production was observed in mice immunized with OVA-liposome made using liposomes that did not contain cholesterol, except in the case of “palmitoyl” liposomes (Figure 3b). Cytokine Production by Splenic CD4+ T Cells of Mice Immunized with OVA-Liposome. Splenic CD4+ cells were taken from mice in the previous experimental groups (Figure 3), and in vitro cytokine production was investigated. Because no cytokine production was observed in the absence of OVA under the culture conditions described in Materials and Methods, data shown in Figure 4 is considered to represent antigen-specific cytokine production. Significant levels of IL-4 production were observed in all groups of mice, and no correlation was found between in vitro T cell IL-4 production and in vivo anti-OVA IgE antibody production (Figure 3b). Similar results were obtained when production of IL-13 was monitored (data not shown). DISCUSSION
In the present study, liposomes with five different cholesterol inclusions (Table 1) were used to make OVAliposome conjugates, and induction of antigen-specific antibody production was monitored. All of the conjugates induced IgE-selective unresponsiveness. Among liposomes with five different cholesterol inclusions, membrane fluidity changed relative to the amount of cholesterol contained (Figure 1). The induction of IgG antibody production by OVA-liposome conjugates prepared using these liposomes correlated well with the membrane
Figure 3. Anti-OVA antibody production in mice immunized with OVA-liposome made using liposomes with four different lipid formulations with (0) or without (9) cholesterol. Six weeks after primary immunization, mice were bled from the tail vein, and serum anti-OVA antibodies were measured: (a) IgG; (b) IgE. Data represent mean and SE of five mice per group. (*) Significant (p < 0.01) difference as compared with liposomes of the same formulation containing cholesterol. ND, not detected.
748 Bioconjugate Chem., Vol. 13, No. 4, 2002
Figure 4. OVA-specific cytokine production by splenic CD4+ T cells of mice immunized with OVA-liposome. Splenic CD4+ T cells in the experiment shown in Figure 3 were stimulated with OVA as described in Materials and Methods, and IL-4 in the culture supernatants was monitored. Data represent mean IL-4 (pg/mL) and SE of five mice per group.
fluidity or with cholesterol inclusion in liposomes; OVAliposome conjugates prepared using liposome that contained no cholesterol and possessed the lowest membrane mobility induced the highest level of anti-OVA IgG antibody production (Figure 2). The results obtained in the present study differ from those obtained in the previous study (12); in the previous study, among liposomes with four different lipid components, the one possessing the highest membrane mobility displayed the highest adjuvant effect for the induction of IgG antibody production when cholesterol was included in all liposome preparations. We then prepared the same panel of liposomes as those used in the previous study in the presence or absence of cholesterol (Table 2) and investigated the induction of anti-OVA antibody production by OVA-liposome conjugates prepared using these liposome preparations. The results obtained using cholesterol-containing liposomes well-confirmed the results of the previous study (Figure 3a). On the other hand, in the cases of “myristoyl”, “palmitoyl”, and “stearoyl” liposomes, liposomes containing no cholesterol displayed significantly higher adjuvant effects than identically formulated liposomes containing cholesterol. Unexpectedly, OVA-liposome conjugates made using liposomes containing no cholesterol induced a significant production of anti-OVA IgE, except in the “palmitoyl” liposome group (Figure 3b). It is unlikely that IgE production was induced in those groups in relation to the induction of high titers of IgG antibody production, because in mice immunized with OVA-liposome prepared using “oleoyl” liposomes, IgE antibody production was induced only in the group of no-cholesterol liposomes, although a similar level of IgG antibody production was induced in both groups, regardless of the presence or absence of cholesterol in liposomes. Interestingly, among liposomes without cholesterol, IgE antibody production was not induced only in the group of mice immunized with “palmitoyl” liposomes. The results were in agreement with those shown in Figure 2; the liposome consisted mainly of palmitic acid (Table 1), and IgE production was not induced regardless of the inclusion of cholesterol. Although, at present, the mechanism of the induction of IgE-selective unresponsiveness by antigen-liposome con-
Nakano et al.
jugates remains unclear, the differential induction of IgE antibody production by OVA-liposome conjugates among liposomes with different lipid formulations, in the absence of cholesterol, could provide an insight into the mechanism. By the inclusion of cholesterol, liposomes are made more resistant to disintegration and biological degradation (7). Perhaps the change in the stability of liposomes caused by cholesterol inclusion affects both adjuvanticity and the capacity to induce IgE-selective unresponsiveness by antigen-coupled liposomes. Both IL-4 and IL-13 are known to play a key role in the induction of IgE antibody production (15). However, CD4+ T cells of mice immunized with OVA-liposome produced IL-4 upon in vitro stimulation with OVA (Figure 4), regardless of in vivo IgE production (Figure 3b), showing that antigen-specific IL-4 production by T cells did not participate in the regulation of IgE production in mice immunized with OVA-liposome. Thus, in the present study, liposomes with different cholesterol inclusions were used to prepare OVA-liposome conjugates, and the induction of anti-OVA antibody production was investigated in mice. The liposomal adjuvanticity for IgG and IgE antibody production was strikingly different among liposomes with different cholesterol inclusions. These results suggest that the cholesterol inclusion in liposomes affects adjuvanticity for IgG production and exerts regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes. ACKNOWLEDGMENT
This work was supported in part by a grant from The Japan Health Sciences Foundation (Research on Health Sciences focusing on Drug Innovation). LITERATURE CITED (1) Audibert, F. M., and Lise, D. (1993) Adjuvants: current status, clinical perspectives and future prospects. Immunol. Today 14, 281-284. (2) Mark, A., Bjorksten, B., and Granstrom, M. (1995) Immunoglobulin E responses to diphtheria and tetanus toxoids after booster with aluminum-adsorbed and fluid DT-vaccines. Vaccine 13, 669-673. (3) Gupta, R. K., and Siber, G. R. (1995) Adjuvants for human vaccinesscurrent status, problems and future prospects. Vaccine 13, 1263-1276. (4) Wassef, N. M., Alving, C. R., and Richards, R. L. (1994) Liposomes as carriers for vaccines. ImmunoMethods 4, 217222. (5) Gregoriadis, G. (1994) Liposomes as immunoadjuvants and vaccine carriers: antigen entrapment. ImmunoMethods 4, 210-216. (6) Gluck, R. (1995) Liposomal presentation of antigens for human vaccines. Pharmacol. Biotechnol. 6, 325-345. (7) Green, S., Fortier, J. D., Madsen, J., Swartz, G., Einck, L., Gubish, E., and Nacy, C. (1995) Liposomal vaccines, in Immunology of Proteins and Peptides VIII (M. Z. Atassi, and G. S. Bixler, Eds.), pp 83-92, Plenum Press, New York. (8) Alving, C. R., Koulchin, V., Glenn, G. M., and Rao, M. (1995) Liposomes as carriers of peptide antigens: induction of antibodies and cytotoxic T lymphocytes to conjugated and unconjugated peptides. Immunol. Rev. 145, 5-31. (9) Fortin, A., Shaum, E., Krzystyniak, K., and Therien, HM. (1996) Differential activation of cell-mediated immune functions by encapsulated and surface-linked liposomal antigens. Cell Immunol. 169, 208-217. (10) Naito, S., Horino, A., Nakayama, M., Nakano, Y., Nagai, T., Mizuguch, J., Komuro, K., and Uchida, T. (1996) Ovalbumin-liposome conjugate induces IgG but not IgE antibody production. Int. Arch. Allergy Immunol. 109, 223-228.
Differential Adjuvanticity in Lipsomes (11) Nakano, Y., Mori, M., Nishinohara, S., Takita, Y., Naito, S., Horino, A., Kato, H., Taneichi, M., Ami, Y., Suzaki, Y., Komuro, K., and Uchida, T. (1999) Antigen-specific, IgE selective unresponsiveness induced by antigen-liposome conjugates. Int. Arch. Allergy Immunol. 120, 199-208. (12) Nakano, Y., Mori, M., Nishinohara, S., Takita, Y., Naito, S., Kato, H., Taneichi, M., Komuro, K., and Uchida, T. (2001) Surface-linked liposomal antigen induces IgE-selective unresponsiveness regardless of the lipid components of liposomes. Bioconjugate Chem. 12, 391-395. (13) Hashimoto, K., Inoue, K., Nojima, S., Tadakuma, T., and Yasuda, T. (1982) Immunogenicity of liposomal model mem-
Bioconjugate Chem., Vol. 13, No. 4, 2002 749 branes sensitized with spin-labeled haptens and topographical distribution of haptens on the membranes. J. Biochem. 92, 1813-1821. (14) Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976) Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 1. Single component phosphatidylcholine liposomes. Biochemistry 5, 4521-4528. (15) Corry, D. B., and Kheradmand, F. (1999) Induction and regulation of the IgE response. Nature 402, B18-B23.
BC0155667
