Liposomes reportedly accumulate in monophagocytic systems (MPSs), such as those of the spleen. Accumulation of considerable amounts of liposome in a MPS can affect immunologic response. While developing a liposomal oxygen carrier containing human hemoglobin vesicle (HbV), we identified its suppressive effect on the proliferation of rat splenic T cells. The aim of this study was to elucidate the mechanism underlying that phenomenon and its effect on both local and systemic immune response. For this study, we infused HbV intravenously at a volume of 20% of whole blood or empty liposomes into rats, removed their spleens, and evaluated T cell responses to concanavalin A (Con A) or keyhole limpet hemocyanin (KLH) by measuring the amount of [3H]thymidine incorporated into DNA. Cells that phagocytized liposomal particles were sorted using flow cytometry and analyzed. Serum anti-KLH antibody was measured after immunizing rats with KLH. Results showed that T cell proliferation in response to Con A or KLH was inhibited from 6 h to 3 days after the liposome injection. Direct cell-to-cell contact was necessary for the suppression. Both inducible nitric-oxide synthase and arginase inhibitors restored T cell proliferation to some degree. The suppression abated 7 days later. Cells that trapped vesicles were responsible for the suppression. Most expressed CD11b/c but lacked class II molecules. However, the primary antibody response to KLH was unaffected. We conclude that the phagocytosis of the large load of liposomal particles by rat CD11b/c+, class II immature monocytes temporarily renders them highly immunosuppressive, but the systemic immune response was unaffected.
A liposome is a lipid particle that is widely used as a drug vehicle in clinical settings and as an adjuvant or delivery system for vaccine antigens. For example, the delivery of Ag inside lipid vesicles is expected to enhance its uptake by antigen-presenting cells such as macrophages and dendritic cells (Dal Monte and Szoka, 1989; Guéry et al., 1996; Dupuis et al., 2001), thereby augmenting the immune response. However, some macrophages act as immune suppressor cells (suppressor macrophages) under certain pathological conditions; the production of nitric oxide (NO) is reportedly involved in that suppression activity (Albina et al., 1991; al-Ramadi et al., 1991; Schleifer and Mansfield, 1993; Dasgupta et al., 1999).
The monophagocytic system (MPS) includes various cells, monocytes, and macrophages capable of phagocytizing particles (Randolph et al., 1999; Dupuis et al., 2001). Because liposomes are particulate, they accumulate in the MPS present in the liver, spleen, bone marrow, and other tissues when injected intravenously into experimental animals (Torchilin, 2005). Therefore, accumulation of liposome in monocyte/macrophages can negatively affect local immune function. Nevertheless, no such effect has been reported to date, possibly because the amount of infused liposome is usually small.
Artificial red blood cells (artificial oxygen carriers) are classifiable into two major types: cell-free and cellular (Ajisaka and Iwashita, 1980; DeVenuto and Zegna, 1983; Chatterjee et al., 1986; Natanson et al., 2008). The latter include hemoglobin molecules encapsulated by liposomes, the major component of which is dipalmitoyl phosphatidylcholine (DPPC) and designated as hemoglobin vesicles (HbVs) (Djordjevich and Miller, 1980; Sakai et al., 1997; Phillips et al., 1999; Chang, 2004). In fact, HbVs have functioned well as blood substitutes in animal models with no noteworthy adverse reactions either in vivo (Sakai et al., 2004; Yoshizu et al., 2004; Cabrales et al., 2005) or in vitro (Ito et al., 2001; Wakamoto et al., 2005; Abe et al., 2006). They also reportedly accumulate in components of the MPS soon after their administration (Sou et al., 2005).
The amount of HbV to be infused as a blood substitute is quite large. Therefore, the negative effect of the liposome on immunological functions might be amplified and easily detected.
In fact, we recently found splenic T cell proliferation to be temporarily but dramatically suppressed after massive administration of HbV into rats. Considering that many trials of cancer immunotherapy have used liposomes containing DPPC, the mechanism behind this immune suppression should be elucidated. In this study, we identified the cells responsible for this phenomenon, elucidated the mechanism behind it, and assessed its effect on both local and systemic immune response. These results might contribute to the progress not only of a liposome-based cancer vaccine strategy but also to progressive development of artificial oxygen carriers such as HbVs.
Materials and Methods
Preparation of HbV and Liposome Suspension.
The HbVs were prepared as described previously (Sakai et al., 1997; Sou et al., 2003). In brief, a hemoglobin solution prepared from outdated red blood cells was heated under a CO gas atmosphere to inactivate any contaminating virus (Abe et al., 2001). After centrifugation and filtration, the hemoglobin solution was mixed with lipids and then extruded through membrane filters with a 0.22-μm pore size to produce liposomes. The lipid composition (molar ratio) was as follows: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/cholesterol (CHOL)/1,5-O- dihexadecyl-N-succinyl-l-glutamate (DHSG)/polyethylene glycol-conjugated 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (PEG5000-DSPE) = 5:5:1:0.033. The mean particle size was 250 nm. All lipids were purchased from Nippon Fine Chemical Co. Ltd. (Osaka, Japan) except PEG5000-DSPE (NOF Corp., Tokyo, Japan). The HbVs were suspended in normal saline; the suspension contained 10 g of hemoglobin/dl, 5.7 g of lipid/dl, and <0.1 endotoxin unit of lipopolysaccharide/ml. Empty vesicles (EVs), which consisted of the same lipid composition as HbV without hemoglobin encapsulation, were also prepared. Liposomes that were composed solely of DPPC (designated DPPC-liposomes) were prepared.
Preparation of FITC-Labeled Empty Vesicle (FITC-Liposome).
Using mixed lipids of DPPC, CHOL, DHSG, and PEG-DSPE (5:5:1:0.033, molar ratio) including 0.1 mol% of N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine and triethylammonium salt (FITC-lipid; Molecular Probes, Carlsbad, CA), FITC-labeled empty vesicles, designated FITC-liposome, were prepared. The mixed lipid powder was hydrated with saline solution at 7 g/dL. The dispersion was introduced into an extruder (Lipex Biomembrane Inc., Vancouver, Canada) and extruded through the membrane filters (final pore size, 0.22 μm, Durapore; Nihon Millipore Ltd., Tokyo, Japan) under pressure using nitrogen gas to obtain FITC-liposome with 233-nm mean diameter.
Animals and Injection of HbV (or Other Liposomes).
Male WKAH rats, 8 to 12 weeks old and weighing 220 to 300 g, were purchased from Japan SLC Inc. (Shizuoka, Japan). Under ether anesthesia, HbVs (or other liposomes) were infused intravenously into the tail at the top load. Saline was infused as a control. The injection volume was 20% of whole blood volume, which was estimated as 56 ml · kg−1 of body weight in rats. This is consistent with approximately 1 liter of whole blood in a 70-kg male human, an amount that is likely to occur in a clinical setting. After the injection, the spleen or lymph node was excised aseptically under ether anesthesia. Then, a single-cell suspension was prepared. Erythrocytes were depleted using red blood cell lysing buffer (IBL, Hamburg, Germany). Then spleen cells were washed in RPMI medium 1640 containing 10% fetal calf serum (FCS). The Executive Review Board (functioning as an Institutional Review Board) of the Hokkaido Red Cross Blood Center reviewed and approved this study's protocol.
Preimmunization with Keyhole Limpet Hemocyanin.
To generate keyhole limpet hemocyanin (KLH)-specific T cells, KLH solution (200 μg of KLH in 0.5 ml of saline) or saline was mixed with the same volume of incomplete Freund's adjuvant. Then 0.5 ml of the mixture (100 μg of KLH) was injected subcutaneously. The HbV or saline was injected 7 days after injection of KLH because preliminary experiment showed that induction of KLH-specific T cell response in the spleen was achieved by 7 days after injection of KLH. Subsequently, the spleen was excised at 6 h, 1 day, 3 days, and 7 days, and the proliferative response of KLH-specific splenic T cells was assayed in vitro.
Assay of the Proliferation of Splenic T Cells in Response to Con A or KLH.
Single spleen cell suspensions in RPMI medium 1640 supplemented with 10% FCS and mercaptoethanol (50 μM) were plated in 96-well plates in a volume of 0.2 ml/well. The cells were cultured in triplicate for 72 h in the presence of 0.3 and 3 μg/ml of Con A (Sigma-Aldrich, St. Louis, MO) or KLH (30 μg/ml) and pulsed with [3H]thymidine (18.5 kBq) for the final 18 h of incubation. Phytohemagglutinin M (PHA-M) was also used as a T cell mitogen in some experiments. Subsequently, the cells were harvested onto glass fiber paper. Radioactivity was measured using a liquid scintillation counter (LS5000 TD; Beckman Coulter, Fullerton, CA). For some experiments, control spleen cells (1 × 105) were mixed with HbV or EV-loaded spleen cells (1 × 105), which were taken from rat loaded with HbV or EV 24 h before, and plated in 96-well plates in a volume of 0.2 ml/well.
Assay of Nitric Oxide and IL-2 Production.
The production of NO in the culture supernatant after 48-h incubation in the presence of Con A (0.3 μg/ml) was measured as the concentration of nitrite using a Griess Assay kit (R&D Systems, Minneapolis, MN). The amount of IL-2 was also measured using the rat IL-2 Immunoassay Quantikine kit (R&D Systems).
Evaluation of T Cell-Suppressive Effect of Liposome-Phagocytized Cells.
Cells that were positive for FITC were sorted from FITC-liposome-loaded splenocytes using FCM (Aria; BD Biosciences, San Jose, CA). Their suppressive effect on the proliferation of Con A-stimulated bulk splenocytes was assayed.
Analysis of Cell Surface Markers and Cell Sorting.
Cell surface markers were analyzed using an LSR flow cytometer (BD Biosciences). The antibodies used for the analysis were allophycocyanin-conjugated CD11b/c (OX42) and phycoerythrin-conjugated anti-class II (OX6), CD80, CD86, CD8a (OX8), CD25 (OX39), CD11a (WT-1), CD172 (OX41), HIS48 (anti-granulocytes), CD103 (OX62), and anti-CD161 (NKR-P1A), and FITC-conjugated anti-rat CD25, all of which were purchased from BD Biosciences. In addition, allophycocyanin-conjugated anti-rat CD3 was purchased from Immunotech (Marseille, France). For each analysis, at least 10,000 events were collected and analyzed using Cellquest software (BD Biosciences).
Detection of Cells with Intracytoplasmic iNOS Protein.
To detect intracytoplasmic inducible NO synthase (iNOS), cells were fixed using FACS Lysing Solution (BD Biosciences) and permeabilized using PBS containing 0.1% saponin and 1% FCS. Then they were stained with mouse FITC-conjugated anti-rat CD11b (BD Biosciences) and phycoerythrin-conjugated anti-rat NOS2 (BioLegend, San Diego, CA).
In some experiments, splenocytes were spun on slides with Cytospin (Thermo Fisher Scientific, Waltham, MA) and stained with May-Grunwald-Giemsa dye (Merck, Darmstadt, Germany). Alternatively, the spleen was fixed with formalin and stained with hematoxylin (Sigma-Aldrich) and eosin (Merck Diagnostica, West Point, PA), respectively, then observed under a light microscope (BX50; Olympus, Tokyo, Japan). Microscopic images were captured using a digital camera (MP5Mc/OL; Olympus) and processed using Win Roof ver. 5.5 software (Mitani Corp., Tokyo, Japan).
Evaluation of the Effect of l-NMMA and nor-NOHA on the Suppression of Splenocyte Proliferation.
For some experiments, an iNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA) (2 mM; Alexis Corp., San Diego, CA), or arginase inhibitor, N-ω-hydroxy-nor-l-arginine (nor-NOHA) (0.5 mM; Calbiochem, San Diego, CA), or both were added to the culture at final concentrations of 2 and 0.5 mM, respectively.
Control bulk splenocytes were washed twice with 1% FCS/PBS and resuspended in the same solution at a concentration of 1 × 107/ml. Subsequently, the cells were incubated with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) at 37°C for 5 min; they were then washed and resuspended in 10% FCS/RPMI medium 1640. The HbV-loaded splenocytes (4 × 105) were placed in the upper chamber (Falcon Transwell, 0.4-μm pore size; BD Biosciences), and CFSE-stained control splenocytes (4 × 105) were placed in the lower chamber (Falcon culture plate). Then they were incubated in the presence of Con A (0.3 μg/ml). At the same time, only the control splenocytes (4 × 105) or a mixture of both vesicle-loaded and control splenocytes were cultured in the lower chamber in the presence of Con A (0.3 μg/ml). The fluorescence intensity of CFSE-stained cells was analyzed using FCM 72 h later.
Evaluation of the Effect of HbV on Primary Antibody Response.
Rats were infused with HbV or saline 6 h before the intravenous injection of KLH (100 μg) on day 0. Peripheral blood was taken from the tail vein on days 5, 7, 10, and 14. Subsequently, serum was separated and stored at −80°C until anti-KLH antibody was measured.
The serum concentration of anti-KLH IgG was measured via enzyme immunoassay. Ninety-six-well microtiter plates (Nalge Nunc International, Rochester, NY) were coated with 0.5 μg of KLH in 100 μl of PBS per well and incubated overnight at 4°C. Plates were washed with PBS once and blocked with 5% dry skim milk in PBS. After incubation for 2 h at room temperature, plates were washed three times with PBS-0.1% polyoxyethylene sorbitan monolaurate (Tween 20). Rat sera were added at a concentration of 1:1000 40% FCS/0.05% Tween 20/PBS. Appropriately diluted standard rat anti-KLH IgG2a (BD Biosciences) in 40% FCS/0.05% Tween 20/PBS was also added to the appropriate plates. Plates were incubated for 60 min and washed three times. Then 100 μl of horseradish peroxidase-conjugated goat anti-rat IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added at a concentration of 1:10,000 in 0.05% skim milk/0.1% Tween 20/PBS to the appropriate plates and incubated for 60 min. Plates were washed three times, then 100 μl of the 3,3′,5,5′,-tetramethylbenzidine one-step substrate system (Dako Japan, Inc., Tokyo, Japan) was added to all wells. Plates were incubated for 15 min and read at an optical density of 450 nm.
Experimental differences from the controls were assessed using two-sided unpaired Student's t tests. Software was used for statistical analyses (ystat2004; Igaku Tosho Press, Co. Ltd., Tokyo, Japan). Values of p < 0.05 were inferred as significant.
Both HbV and Empty Vesicles Suppressed the Proliferative Response of Splenic T Cells to Con A.
The proliferative response of both EV- and HbV-loaded splenic T cells was clearly inhibited at a lower dose of Con A (0.3 μg/ml) than that of saline-loaded (control) splenic T cells (p < 0.01) (Fig. 1a). This result was quite reproducible. However, at a high dose of Con A (3.0 μg/ml), the inhibition was mild. Therefore, the concentration of Con A used in the subsequent experiment was fixed at 0.3 μg/ml. As with splenocytes, the Con A-induced proliferation of HbV-loaded lymph node cells was also decreased at 0.3 μg/ml of Con A (p < 0.05) compared with that of control lymph node cells (Fig. 1b). In addition, the suppressive effect of EV tended to be stronger than that of HbV. Furthermore, PHA-M induced T cell proliferation was suppressed at 2 to 8 μg/ml of PHA-M (data not shown).
Kinetics of the HbV-Induced Suppression of the T Cell Proliferative Response to Con A and KLH.
Suppression was observed clearly from 6 h to 3 days after the injection. It disappeared completely 7 days after the injection (Fig. 1c). The KLH-specific proliferation of splenic T cells was also inhibited by the HbV infusion, the kinetics being the same as that for Con A stimulation.
IL-2 and NO Were Detected in the Supernatant Cultured for 48 h in the Presence of Con A.
Although the T cell proliferation of HbV-loaded splenocytes was inhibited reproducibly at 0.3 μg/ml of Con A, the amount of IL-2 in the supernatant of these splenocytes was comparable with that produced by saline-loaded splenocytes (Fig. 2a). Moreover, flow cytometric analysis revealed that the expression of high-affinity IL-2 receptor on the surface of the former T cells was comparable with that of the latter (data not shown). These data suggest that T cells were activated normally. Furthermore, NO was shown to be produced by both HbV-loaded and saline-loaded splenocytes, irrespective of the presence of Con A. However, the production of NO in HbV-loaded splenocyte culture supernatant tended to be higher than that of saline-loaded splenocyte culture supernatant (Fig. 2b).
T Cell Proliferation Was Restored by l-NMMA or nor-NOHA.
Through the preliminary experiment, we confirmed that l-NMMA (iNOS inhibitor) (2 mM) and nor-NOHA (arginase inhibitor) (0.5 mM) used in the experiment did not influence the proliferation of Con A-stimulated saline-loaded T cells at all. Actually, l-NMMA(2 mM) was sufficient to inhibit the production of NO produced by Con A (0.3 μg/ml) stimulation, although nor-NOHA did not influence the production of NO at all (Fig. 2b). Both l-NMMA and nor-NOHA restored T cell proliferation to some degree, with better restoration by l-NMMA (Fig. 2c). These data show that NO was involved in the suppression of T cell proliferation.
Cell-to-Cell Contact Is Necessary for Suppression.
When CFSE-stained control splenocytes were stimulated with Con A in the presence of HbV-loaded splenocytes, no cell division was observed (Fig. 2d, bottom). In contrast, when they were separated from HbV-loaded splenocytes using a transwell, considerable cell division was observed (Fig. 2d, middle).
Identification of Cells Responsible for T Cell Suppression.
When control spleen cells (1 × 105) were stimulated using Con A (0.3 μg/ml) in the presence of spleen cells (1 × 105) loaded with HbV (or EV) 24 h prior, T cell proliferation was suppressed (Fig. 3a). Furthermore, cells that had phagocytized FITC-liposomes (FITC-positive cells) (Fig. 3b) inhibited the proliferation of control splenic T cells dose-dependently (Fig. 3c). In addition, FITC-negative cells proliferated well compared with FITC-EV-loaded bulk splenocytes. They proliferated even better than control splenocytes at 1 μg/ml of Con A (Fig. 3d).
Phenotypic Analysis of Cells Responsible for T Cell Suppression.
The FITC-positive cells were gated first. These cells accounted for approximately 5% of all splenocytes. Then, the phenotype of those cells was analyzed. Most were positive for CD11b/c, but negative for class II molecules, CD80, or CD86 (Fig. 4a, top). In addition, they were weakly positive for CD4 and negative for CD3, CD25, CD8, and CD103. Some (14%) were positive for both CD11b/c and HIS48 (data not shown).
Microscopic examination revealed that unique cells appeared after the injection of vesicles. They were larger than lymphocytes. Moreover, they had cytoplasm that was abundant in vesicle-like particles and had a nucleus with an irregular rim (Fig. 4b).
Flow cytometric analyses of iNOS-positive cells revealed that the percentage of iNOS-positive cells among CD11b-positive cells is not necessarily higher in HbV-loaded splenocytes than in saline-loaded splenocytes (data not shown), suggesting HbV load does not increase in the number of NO-producing phagocytes.
Effect of HbV Infusion on Primary Antibody Response.
Elevation of anti-KLH IgG antibody after the first challenge of KLH was demonstrated in both HbV-loaded and control rats, with no apparent difference between them (Fig. 5).
DPPC-Liposomes Can Induce Immune Suppression.
Macroscopic examination revealed that, like HbV and empty vesicles, DPPC-liposome accumulated in the spleen (Fig. 6a). Subsequent histological examination revealed unique cells with abundant cytoplasm (Fig. 6b). Moreover, DPPC-liposomes induced suppression of T cell proliferation (Fig. 6c).
We showed that the infusion of HbV temporarily suppressed not only mitogen-induced but also antigen-induced T cell proliferation in rat splenocytes (Fig. 1, a and c), which indicated that HbV infusion suppressed T cell receptor-mediated T cell proliferation.
The Hb molecule can be ruled out as a cause of suppression because even empty vesicles, HbV without Hb molecules in them, induced suppression.
Among the components of HbV, DPPC was responsible for the suppression. Similarly to HbV and EV, DPPC-liposomes, which contain no CHOL, DHSG, PEG, or Hb, were shown to accumulate in the spleen and induce suppression (Fig. 6). Whether CHOL, DHSG, and PEG are involved in the suppression or not remains unknown at present. However, considering that the suppressive effect of DPPC-liposome is more prominent than that of HbV, it is less likely that these components play a principal role in the suppression.
According to a previous report (Sakai et al., 2001), HbV began to accumulate in monocyte/macrophages present in the red pulp of the spleen. Then they were observed in considerable amounts on day 3, before gradually disappearing by day 7. This pattern resembled that of immune suppression induced by the infusion of HbV (Fig. 1c). Consequently, the accumulation of liposomal particles in the spleen must be related somehow with this temporary immune suppression. Actually, splenocytes acquired the ability to inhibit the proliferation of control splenocytes after the injection of HbV (or EV) (Fig. 3a). Moreover, cells that trap liposomal vesicles (FITC-liposome) were shown to be responsible for immune suppression (Fig. 3,b and c). The result that cells sorted as FITC-negative cells acquired greater proliferative capacity might support this inference (Fig. 3d). Therefore, it was concluded that the cells responsible for the immune suppression were phagocytic cells. The expression of CD11b/c, which is a hallmark of macrophage/monocyte lineage, was consistent with the conclusion. Furthermore, their lack of class II molecule, CD80, and CD86 expression, coupled with their morphology (Fig. 4, a and c), indicated that they are immature monocytic cells. It is possible that they lost these molecules when phagocytizing liposomes. However, this might not be the case because, even in control splenocytes, most CD11b/c+ cells were negative for class II molecules, CD80, and CD86 (data not shown).
Based on the results obtained from a series of experiments conducted to elucidate the mechanism for immune suppression, direct cell-to-cell contact (Fig. 2c) is necessary, and both NO and arginase are involved (Fig. 2d), with a more principal role played by the former.
Phagocytosis of HbV might not be linked to the generation of NO, because the percentage of iNOS-positive cells among CD11b-positive cells (macrophage lineage) was not necessarily higher in HbV-loaded splenocytes than in saline-loaded splenocytes (data not shown). It is unclear why NO generated by the former tended to be higher than that generated by the latter. However, it is possible that phagocytosis of HbV might result in enhancing the generation of NO.
Referring to T cells, based on data about IL-2 secretion (Fig. 2a) and CD25 expression, HbV-loaded splenic T cells were activated in a normal way. However, they were unable to proliferate. It must be emphasized that these mechanisms and T cell status closely resemble those of T cell suppression caused by myeloid-derived suppressor cells (Mazzoni et al., 2002; Ostrand-Rosenberg and Sinha, 2009), although most liposome-phagocytizing cells did not express HIS 48 antigen, which is reportedly a marker of rat myeloid-derived suppressor cells (Dugast et al., 2008).
As described previously, the suppressive effect of HbV tended to be milder than that of EV (Fig. 1a). The Hb molecule might be responsible for the difference because the only difference between them was the presence of HbV. It is possible that NO is trapped by Hb molecules, engendering the decrease of its immunosuppressive effect. This trapping phenomenon might also explain why the suppressive effect of DPPC-liposome is more prominent than that of HbV (Fig. 6c).
Whether HbV infusion induced systemic immune suppression or not is the next concern of this research. The antibody response to KLH depends on T cells. Therefore, the primary antibody response to KLH was evaluated as a systemic immune response. The primary antibody response was unaffected by infusion of HbV, at least in our experimental conditions (Fig. 5). Therefore, it is less likely that massive infusion of HbV induces severe immunosuppressive effect on the host. The findings that T cell suppression was a transient phenomenon and that immune suppression in the lymph node cells was rather milder than that of splenocytes (Fig. 1b) support this. However, this finding must be emphasized: transient induction of immunosuppressive activity in HbV-phagocytizing cells is unique. Considering that infusion of HbV induces no strong adverse reaction, HbV (or liposomes) might be preferred as an immunosuppressive agent in certain clinical settings.
In conclusion, we demonstrated the existence of a subset of CD11b/c+, class II immature monocytes in rat spleen that can swiftly and transiently acquire strong T cell-suppressive potential via the phagocytosis of a considerable amount of HbV or DPPC-liposomes. Direct cell-to-cell contact and both iNOS and arginase are involved in that suppression. Immune suppression might be restricted to a local site such as the spleen, which has abundant phagocytic cells.
Participated in research design: Takahashi, Azuma, Sakai, Sou, and Fujihara.
Conducted experiments: Takahashi, Azuma, Wakita, and Abe.
Contributed new reagents or analytic tools: Sakai and Sou.
Performed data analysis: Takahashi, Azuma, Fujihara, Horinouchi, Nishimura, and Ikeda.
Wrote or contributed to the writing of the manuscript: Takahashi, Azuma, and Sakai.
Other: Horinouch and Kobayashi acquired funding for the research.
We dedicate this article to the late Emeritus Professor Eishun Tsuchida, Research Institute for Science and Engineering, Waseda University, Tokyo, Japan.
This work was supported in part by the Ministry of Health, Labor, and Welfare of Japan, Health Sciences Research Grants, and Research on Public Essential Drugs and Medical Devices [Grant H18-Soyaku-Ippan-022].
H.S. and K.S. are inventors listed in patents related to the production and utilization of hemoglobin vesicles.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- nitric oxide
- inducible NO synthase
- monophagocytic system
- hemoglobin vesicle
- empty vesicle
- keyhole limpet hemocyanin
- dipalmitoyl phosphatidylcholine
- 1,5-O- dihexadecyl-N-succinyl-l-glutamate
- carboxyfluorescein diacetate succinimidyl ester
- fetal calf serum
- fluorescein isothiocyanate
- flow cytometry
- Con A
- concanavalin A
- polyethylene glycol
- phytohemagglutinin M
- phosphate-buffered saline.
- Received July 8, 2010.
- Accepted January 5, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics