Artificial Organs

36(2):194201, Wiley Periodicals, Inc.

2011, Copyright the Authors
Artificial Organs 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Effect of Liposome-Encapsulated Hemoglobin on

Antigen-Presenting Cells in Mice
*Akira T. Kawaguchi, *Junko Aokawa, Yuko Yamada, *Fumiaki Yoshiba, *Shunichi Kato,
and Yoshie Kametani
Departments of *Cell Transplantation and Regenerative Medicine and Immunology, Tokai University School of Medicine,
Isehara, Kanagawa, Japan

Abstract: Liposome-encapsulated hemoglobin (LEH) is

removed from the circulation and degraded in the reticuloendothelial system, including dendritic cells (DCs) and
macrophages. Therefore, LEH at a large dose may overload the system, cause a competitive inhibition in antigenpresenting activity, and impair the immune response of
the host. Changes in cellularity of immunocompetent cells
were monitored serially up to 4 weeks by flow cytometry
in wild-type mice receiving 20 mL/kg of LEH, syngeneic
red blood cells (RBCs), or saline. DCs were collected
from the host spleen 1, 7, and 28 days after receiving the
solution and were cocultured with nave cluster of differentiation 4 T cells from T-cell receptor transgenic mice in
the absence or presence of third-party antigens. After

LEH administration, the cellularity of DCs and macrophages in the recipient spleen remained unchanged from
control mice receiving RBCs or saline. While subset populations and costimulatory molecule expressions were different, DCs from LEH-administered mice expressed high
levels of interleukin-2 production and helper T-cell activation in response to a third-party antigen and superantigens, as did the DCs from control mice receiving RBCs
or saline. The results suggest that 20 mL/kg of LEH does
not greatly alter antigen-presenting activity to third-party
antigens. Key Words: Artificial oxygen carriers
Antigen-presenting cellsDendritic cellsMacrophages
PhagocytosisEmergency transfusionHemorrhagic
shockContaminationImmune response.

Artificial oxygen (O2) carriers have been developed as substitutes of red blood cells (RBCs) for
transfusion (1,2). Although vascular adverse events
observed in cell-free hemoglobin in clinical trials (3)
were reportedly avoided in experimental studies
using liposome-encapsulated hemoglobin (LEH;
TRM-645, Terumo, Tokyo, Japan) (47) or hemoglobin vesicle (HbV) (8,9), there remain concerns
regarding its use as a transfusion substitute because
of a much shorter retention time than that of RBCs.
Circulation half-life (T1/2) of LEH and HbV depends
on species as well as doses; it is approximately 1 day
in rodents (49) and 23 days in primates when

1020 mL/kg is used (10,11). While LEH is removed

from the circulation and disposed of by the reticuloendothelial system (RES) similarly to RBCs, the
possible use of LEH for emergency transfusion
before identical donor blood becomes available (12)
may impose an acute metabolic load of hemoglobin
and lipids on RES or antigen-presenting cells
(APCs), such as dendritic cells (DCs) or macrophages, which plays a pivotal role in the triggering of the
hosts immune defense against third-party antigens.
Thus, this raises a serious concern, as hemorrhagic
shock patients are often exposed to pathogens at
accidents or surgery causing acute blood loss necessitating emergency transfusion (12). In our previous
study (4), a single injection of LEH at a moderate
dose (10 mg/kg) did not largely affect the murine or
human immune status nor did it interfere with the
hosts early response to bacterial enterotoxins (toxic
shock syndrome toxin-1 [TSST-1]), so far as lymphocyte cellularity and interleukin (IL)-2 producibility
were concerned. While Sakai and colleagues (2)
reported that administration of 20 mL/kg of HbV,

doi:10.1111/j.1525-1594.2011.01269.x
Received August 2010; revised March 2011.
Address correspondence and reprint requests to Dr. Akira T.
Kawaguchi, Department of Cell Transplantation and Regenerative
Medicine, Tokai University School of Medicine, Shimokasuya 143,
Isehara, Kanagawa 259-1193, Japan. E-mail: akira@is.icc.u-tokai.
ac.jp

194

aor_1269

194..201

ARTIFICIAL O2 CARRIER AND ANTIGEN-PRESENTING ACTIVITY

195

A
I. Antigen recognition

DC

Helper T cell

APC
MHC
class II

CD3

II. Phagocytosis

MHC class

APC
MHC
class II

CD 25

TCR

*
IL-2

CD4

III. Antigen
presentation
Costimulator

APC

Nucleus
B7

CD28

Costimulator

MHC
class II
Costimulator

B7

C
CD3

MHC
class II

Costimulator

OVA

specific to

OVA

CD4

B7

*
CD 25

TCR

Costimulator

CD28

IL-2

GFP *

OVA-IL-2/GFP mouse helper T cell

BALB/c mice

CD3

MHC
class II

*
CD 25

TCR
specific to

IL-2

OVA
TSST-1
Costimulator

B7

CD28

Costimulator

GFP *

CD4

FIG. 1. Activity of antigen-presenting cells. Antigen-presenting cells (APC, A) recognize foreign material (I), phagocytes (II), and present
as a specific antigen (III) on MHC class II surface markers together with costimulator protein (B7). Helper T cell (B) with a specific receptor
(TCR) comes to be activated through TCR (signal 1) and costimulator protein pathway (signal 2) to activate helper T cell, producing IL-2
and CD25 as a surface marker or IL-2 receptor. Helper T cell (C) from OVA23-3/IL-2-GFP mice can be activated through TCR specific
to OVA and produce GFP in plasma together with IL-2 production. Helper T cells may also be stimulated by TSST-1 without any specific
antigen. Activation of helper T cells from transgenic mice may be evaluated sensitively and specifically by FACS, gated for GFP (*) as well
as CD25 (*).

similar to LEH, transiently reduced phagocytic activity by 40%, we directly examined the activity of APCs
from LEH-administered mice using a system that
allows the mimicking of the initial encounter of these
two cell types with LEH in vivo. In this study, first, we
examined in vivo changes in an immunocompetent
cell population in LEH-treated mice and then the
activation (Fig. 1A) and subpopulation changes in
splenic DCs and macrophages. Finally, the activity of
DCs (Fig. 1B) in the absence or presence of thirdparty antigens was tested in vitro, using cluster of
differentiation (CD)4+ T cells (helper T cells) harvested from newly developed transgenic mice (Fig.
1C) so as to increase the sensitivity and specificity in

detecting the antigen-presenting activity of DCs from

recipient animals.

MATERIALS AND METHODS

Liposome-encapsulated hemoglobin
Encapsulation of hemoglobin has been proven to
be effective in avoiding extravasation and direct
contact of hemoglobin with vascular structure in
LEH (1,47) as well as in HbV (2,8,9). Relevant characteristics of LEH have been reported. Briefly, LEH
is a liposome capsule, measuring 230 nm in mean
diameter and contains human hemoglobin purified
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A.T. KAWAGUCHI ET AL.

from donated blood that was outdated for transfusion (1). The liposome capsule was coated with
polyethylene glycol to reduce aggregation and recognition by RES, as well as to prolong the circulatory
half-life to less than 1 day in mice (4), approximately
1 day in rats (57), and 23 days in nonhuman primates (10,11). In this preparation, inositol hexaphosphate was coencapsulated to control O2 affinity to
P50O2 = 4050 mm Hg in LEH, lower than that of
rodent RBCs (P50O2 = 30 mm Hg). LEH was suspended in saline at a hemoglobin concentration of
6 g/dL or 20% of volume, which has reduced viscosity
(2 cp) compared with blood (5 cp) and specific
gravity close to that of plasma. Phosphate buffered
saline (PBS) and murine syngeneic RBCs, washed
and prepared at a hematocrit of 20%, were used as
control solutions.
Mice
Eight-week-old female wild-type mice (BALB/c)
were purchased from CLEA Japan (Kawasaki, Kanagawa, Japan). T-cell receptor (TCR) transgenic mice
(I-Ad, ovalbumin [OVA]323339 specific) termed
OVA23-3 mice (13), expressing TCR specific for a
peptide fragment of OVA (OVA323339) presented by
the I-Ad class II major histocompatibility complex
(MHC) molecule, were maintained on a BALB/c
background over 16 generations. This OVA23-3
mouse was crossed with transgenic mouse strain carrying 5 flanking region of IL-2 gene conjugated with
enhanced green fluorescent protein (GFP) coding
region (IL-2-GFP mouse, kindly provided by Professor Ellen V. Rothenberg) for at least nine generations
as described previously (14). The offsprings were
determined as transgene carriers by polymerase
chain reaction analysis of genome DNA. The
OVA23-3 transgenic mice, IL-2-GFP mice, and their
offspring (OVA23-3/IL-2-GFP) were kept under specific pathogen-free conditions in the animal facility of
Tokai University School of Medicine (Shimokasuya,
Kanagawa, Japan).
Administration of LEH, RBCs, and PBS
All experiments were approved by the institutional
review board of Tokai University School of Medicine.
Animals received humane care as required. Female
wild-type BALB/c mice (8~10 weeks old) were anesthetized with ether and received a single intravenous
injection of LEH (20 mL/kg), washed syngeneic
RBCs, or PBS via the tail vein on day 0. Syngeneic
RBCs were collected from nontreated nave BALB/c
mice by Ficoll-Conray Lymphosepal (ImmunoBiological Laboratories, Gunma, Japan) and centrifuArtif Organs, Vol. 36, No. 2, 2012

gation (2000 g, 30 min), washed three times with

saline, and diluted with saline to a final RBC suspension, 20% hematocrit.
Monoclonal antibodies and flow cytometry
T cells and B cells were identified by fluorescentactivated cell sorter (FACS) according to their
expressions of CD3 (pan T cell), CD4 (helper T cell),
CD8 (cytotoxic/suppressor T cell), and CD19 (B cell),
respectively. DCs and macrophages were identified
according to their expression of CD11c and CD11b,
respectively. T-cell activation was determined by
CD25 (T-cell activation antigen). B7-1 (CD80) and
B7-2 (CD86) are the most extensively studied
costimulatory molecules of the B7 family (DC activation antigen, Fig. 1A) (15). Interaction of CD28 on
T cells with either CD80 or CD86 has been found to
augment T-cell activation, promote T-cell survival,
and enhance IL-2 production (16) (Fig. 1B). Thus,
antigen-presenting ability may be evaluated by
simply analyzing the expression of these molecules
(B7).
CD4 T cells from OVA23-3/IL-2-GFP mice
CD4 T cells (helper T cells) were isolated from a
single-cell suspension of splenocytes from OVA23-3/
IL-2-GFP mice by positive selection using
anti-mouse CD4-PerCP.Cy5.5 and goat anti-rat
immunoglobulin G microbeads and magnetic cell
sorting system columns (Miltenyi Biotec, Gladbach,
Germany). OVA23-3/IL-2-GFP mice have a single
TCR repertoire (13), enabling the activation of,
ideally, all T cells by a specific antigen, OVA. Moreover, the activation and production of IL-2 of mouse
T cells can be detected by identifying GFP by flow
cytometry (Fig. 1C) instead of measuring IL-2 in the
supernatant (Fig. 1B). In the current study, therefore,
both the IL-2 production and the expression of
CD25, an activation marker, by helper T cells were
detected by flow cytometry without damaging the
cells. These characteristics allow amplified, sensitive,
and specific evaluation of the helper T-cell activation
process. The purity of CD4+ cells isolated by this
method was above 95%, as confirmed by flow
cytometry.
DCs from BALB/c mice 1, 7, and 28 days
after administration
After collecting blood, BALB/c mice were sacrificed 1, 7, and 28 days after intravenous administration, and the whole spleen was excised and weighed.
Spleen cells were isolated by cutting spleens into

ARTIFICIAL O2 CARRIER AND ANTIGEN-PRESENTING ACTIVITY

Cellularity of APCs in spleen

The percentage of total CD11c+ DCs from spleen
did not significantly change until 28 days after LEH
administration (Fig. 2). The prevalence of DCs (Fig.
3A) and their subsets in spleen was analyzed 24 h
after administration, and was expressed as indices to
equivalent changes occurring in PBS-treated mice.
While the value of the DCs index was similar
between LEH and RBC administered to mice after
24 h (Fig. 3A, left), the percentage of CD11c high-cell

B cells

0.5

Pan T cells

Suppressor T cells

0.5

Helper T cells

DCs

MOs

0.5

Statistical analysis
Data are expressed as mean standard error.
The value obtained from LEH- or RBCadministered mouse spleen cells divided by the
comparable value from PBS-treated mice was
shown as mean index standard deviation (SD).
The statistical significance of differences between
two groups was analyzed by Students t-test, and
multiple comparisons were performed using analysis
of variance. A P value less than 0.05 was considered
statistically significant.

Cellularity of immunocompetent cells in

BALB/c mice
The kinetics of the immunocompetent cell ratio
obtained from LEH-treated BALB/c mouse spleens
was assayed by flow cytometry. Raw data were
divided by comparable values from PBS-treated mice
and shown as mean index SD (Fig. 2). The ratio
stayed around 1 in any of the categories of pan T cells
(CD3) and B cells (CD19), helper T cells (CD4), and
cytotoxic T cells (CD8), DCs (CD11c), and macrophages (CD11b), showing no consistent or significant
changes in prevalence 1, 7, 14, and 28 days after
administration of LEH (20 mL/kg), as compared with
the same administered volume of PBS. However, the
total number of spleen cells of LEH-treated mice
increased to 1.8-fold compared with control mice
receiving saline 1 day after LEH administration.

Stimulation
Splenic DCs (1.2 105) purified from BALB/c
mice administered with LEH, RBCs, or PBS were
incubated with CD4 T cells (6 105) from OVA233/IL-2-GFP mice in 48-well flat-bottom plates
in a final volume of 200 mL. These cocultured
cells were maintained at a density of 2.5 106/mL
cells in RPMI, supplemented with 10% heatinactivated FCS (Invitrogen, Carlsbad, CA, USA),
160 mg/mL gentamicin, 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES, SigmaAldrich, St. Louis, MO, USA), 2 mM glutamine, and
50 mM 2-mercaptoethanol at 37C in 5% CO2. Each
well was stimulated with OVA (1 mg/mL, SigmaAldrich), TSST-1 (1 mcg/mL, Toxin Technology, Inc.,
Sarasota, CA, USA), or PBS at 37C for 24 or 48 h.
After the stimulation, the cells were harvested,
stained with anti-CD4-PerCP-Cy5.5, and analyzed
by FACSVantage to check their purity. Cells with
more than 90% purity were used for the assay of
antigen-presenting ability of T-cell activation and
IL-2 production (Fig. 5A); expressions of CD4, GFP,
and CD25 were assessed by flow cytometry in the
lymphoid gate. GFP expression was monitored by
fluorescence 1 channel (Fig. 5B).

RESULTS

LEH / PBS ratio

pieces and digesting them in 5 mL of Collagenase D

(Roche, Basel, Switzerland) at 37C for 30 min. The
cells were washed, the RBCs lysed by incubation for
5 min in ammonium chloride buffer, pH 7.2, washed
again, and resuspended in complete Roswell Park
Memorial Institute medium (RPMI) and 10% fetal
calf serum (FCS). The data were from three experiments, each using pooled spleen cells from three mice
per group. The total number of spleen cells was
counted. CD11c+ DCs obtained from the spleens
of BALB/c mice from the three groups (LEH, RBC,
and PBS) were stained with anti-mouse CD11cfluorescein isothiocyanate (FITC) and anti-FITC
microbeads and then positively isolated by automatic
magnetic cell sorting system. CD11c+ cells, as well as
CD11b+, were gated for subset flow cytometry.

197

14

28

Days after administration

FIG. 2. Cellularity of splenic immunocompetent cells. There was
no significant or consistent change in the percentages of immunocompetent cells; between pan T cell (CD3) and B cell (CD19),
between T cell subsets (CD4 vs. CD8), and between DCs
(CD11c) and macrophages (MOs, CD11b) in spleen analyzed
sequentially using flow cytometry.
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A.T. KAWAGUCHI ET AL.

RBC/

LEH/ PBS

PBS

RBC/

P =0.03

CD11b+ index

1.5
CD11c+ index

LEH/ PBS

PBS

1.5

1.0
0.5

0.5

0.0
CD11c(+)
Total

CD11c(+)
High

CD11c(+)
Intermediate

CD11b (+)
Total

CD11b (+)
High

CD11b (+)
Intermediate

FIG. 3. Percentages of cells expressing CD11c+ and CD11b+. Percentages of CD11c+ cells (DCs, A) as well as CD11b+ cells (MOs, B)
in spleens analyzed 24 h after 20 mL/kg of intravenous administration of LEH, RBC, or PBS. Data were from three experiments, each
containing three mice per group. Asterisk (*) depicts P < 0.05 as compared with RBC-treated group.

fraction (CD11chigh, mainly including myeloid DCs or

conventional DCs) declined significantly to 60% of
the total CD11c+ DCs found in spleens of RBCadministered mice (Fig. 3A, middle). Meanwhile, the
CD11c intermediate cell fraction (CD11cint, mainly
including plasmacytoid DCs) tended to be higher in
the presence of LEH, although the difference did not
achieve significance (P = 0.075). The percentage of
total CD11b+ cells, mostly macrophages, from spleen
did not significantly change 1, 7, and 28 days after
LEH administration (data not shown), although it
tended to be lower in the presence of LEH. Subsets
of macrophages were shown 1 day after administration (Fig. 3B); the percentage of CD11bhigh cell frac-

tion (Fig. 3B, middle) and CD11b intermediate cell

fraction (CD11bint, Fig. 3B, right) tended to be lower
in the presence of LEH, but significance was not
achieved (P = 0.13).

RBC/

1.5

PBS

LEH/ PBS

Expression of costimulatory molecules on

CD11c+ DCs
DCs up-regulated CD86 expression after administration of LEH compared with RBCs (Fig. 4A). The
difference in CD86 expression was significant at
day 1, became less prominent at day 7, and returned
to equivalence 28 days after administration between
the LEH- and RBC-transfused groups. Costimulatory CD86 expression on the CD11chigh subset was

P <0.004

RBC/

PBS

LEH/ PBS

4.0

1.0

CD86 index

CD86 index

5.0

0.5

3.0

P =0.04

2.0
1.0

0.0

0.0

7
Days after administration

28

CD11c(+) High

CD11c(+) Intermediate

FIG. 4. Expression of costimulatory molecules on CD11c+ DCs. Expression of costimulatory molecules (CD86) on spleen DCs (A) 1, 7,
and 28 days after administration of LEH or RBC compared with PBS-treated mice as control. Spleen cells were stained with anti-CD86
and anti-CD11c, and frequency and intensity were measured by FACS gating on CD11c+ cells. Those values in LEH- or RBCadministered mice 24 h after administration were obtained for CD11c high cell subset (CD11chigh) or CD11c intermediate cell subset
(CD11cint) and presented as percentage of CD86-expressing cells (B) using PBS-treated mouse spleens as standard.
Artif Organs, Vol. 36, No. 2, 2012

ARTIFICIAL O2 CARRIER AND ANTIGEN-PRESENTING ACTIVITY

B
DCs coculture with CD4+cells
for 24 & 48 hours with

LEH
RBC
PBS

24h
Splenectomy

0h

PBS

OVA IL-2/GFP
mice

OVA TSST-1

OVA

(-)

TSST-1

4.1

18.5

21

4.2

15.2

18

4.2

16.1

20

LEH

Spleen CD4+
T cells

CD11c+ DCs
Purified by MACS

Administration

BALB/c mice

CD25

199

RBC

PBS

RBC /PBS

LEH /PBS

1.2

GFP (IL-2)

1.0
0.8
0.6
0.4
0.2
0.0

OVA

TSST-1

FIG. 5. DCs from LEH-administered spleen. Three DC populations purified from pooled LEH-, RBC- and PBS-administered mouse
spleens were cocultured with nave CD4 T cells harvested from OVA23-3/IL-2-GFP transgenic mice without (-) or with the presence of
ovalbumin (OVA) or TSST-1 (TSST-1). The expressions of CD4+, GFP+, and CD25+ cells were assessed by flow cytometry in the
lymphoid gate, and the frequency of IL-2/GFP+ cells was assessed 24 h after stimulation (A). Values in the plots represent the proportion
(%) of the CD4+ gate that IL-2-GFP and CD25+ comprise (B). The percentage of dual positive cells (C), average of three experiments,
revealed that DCs from spleens 24 h following LEH administration showed no impairment in their ability to activate helper T cells as
compared with the mice administered with RBC.

highly enhanced (P < 0.01), but the CD86 expression

on CD11cint was significantly attenuated (P < 0.04)
1 day after administration (Fig. 4B). In contrast,
costimulatory CD80 expression on the total CD11c+
DCs transiently declined to 40% of DCs from RBCtransfused mice, while CD11c + CD80 high cells
became predominant in LEH-treated mice compared
with RBC-transfused mice 7 days later (data not
shown).

IL-2 production and CD25 expression as

APC activity
The frequency of GFP+ cells was assessed 24 and
48 h after stimulation with OVA peptide or TSST-1
(Fig. 5A, center) in the coculture of nave CD4+ T
cells from OVA23-3/IL-2-GFP transgenic mice (Fig.
5A, right) incubated with DCs purified from pooled

LEH-, RBC-, and PBS-administered mouse spleens

(Fig. 5A, left). The expressions of CD4, GFP, and
CD25 were assessed by flow cytometry in the lymphoid gate (Fig. 5B). The FACS profiles are representative of coculture from 24 h postantigen stimulation,
presenting the proportion of CD4+ cells comprising
IL-2-GFP (indicating IL-2 expression) and CD25
expression (indicating T-cell activation). In all cases,
1017% of CD4 T cells up-regulated GFP expression
irrespective of the origin of DCs, and 6070% of CD4
T cells up-regulated CD25 in all culture conditions
with antigen stimulation. There was no difference
between coculture durations of 24 and 48 h. In three
experiments, the average of cell population doublepositive for GFP as well as CD25 was similar
between mice infused with RBC or LEH, irrespective
of the presence of OVA or TSST-1 (Fig. 5C). There
was no significant deviation from the ratio 1 or
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A.T. KAWAGUCHI ET AL.

difference between the groups either 7 or 28 days

after infusion (data not shown).
DISCUSSION
Clinical needs for transfusion often derive from
trauma and/or surgery, involving concurrent exposure to pathogens and/or tumor antigens. We have
been using type O Rh (+)-washed and -packed RBCs
for emergency transfusion for patients with hemorrhagic shock before identical RBCs become available
(12). Although a shortened ischemic period has been
demonstrated to be advantageous in terms of survival
over the risk of minor mismatch transfusion (12),
the volume of transfusion derived from a 400-mL
package amounted to 3.05 1.20 units of washed
and packed RBCs. While this amount does not pose
any concern in the case of RBCs, the shorter vascular
retention time of LEH may raise the potential risk
that LEH would overload the RES and attenuate the
antigen-presenting activity of the host. APCs take up
and process antigens and present them to T lymphocytes with the help of MHC molecules as the initial
trigger of adaptive immune response (Fig. 1A),
inducing either tolerance to self-specific peptides or
immunity against foreign antigens (Fig. 1B). In the
current study, the antigen-presenting ability to thirdparty antigens was evaluated in the presence or
absence of LEH 20 mL/kg administration, a dose
equivalent to that of emergency transfusion (12).
We previously reported that 10 mL/kg of LEH
administration induced no obvious change of cellularity in T cell subsets and B cells in murine, as well as
in the reconstituted human immune system in the
first 4 weeks postadministration (4). Also in the
current study after the administration of 20 mL/kg of
LEH (Fig. 2), the population of immunocompetent
cells, including DCs and macrophages, in the peripheral blood or spleen did not differ significantly from
the changes occurring in mice treated with PBS. Splenomegaly was observed in recipient mice transfused
with LEH; the 1.8-fold increase in the total number
of cells in the spleen 1 day after LEH infusion was
comparable with the reported degree of splenomegaly after HbV infusion (2). As the spleen is the
main organ to dispose of LEH or HbV, accumulation
of such foreign materials may induce the migration
of other immunocompetent cells or scavengers,
although a bias of cellularity was not detected
(Fig. 2).
The CD11c expression level is reported to be different between myeloid DCs and plasmacytoid DCs
(17,18). Therefore, it is impossible to clearly identify
which subset of DCs or macrophages was altered
Artif Organs, Vol. 36, No. 2, 2012

between the LEH-infused and the RBC-transfused

mice. While the total cellularity of CD11c+ DCs
remained unchanged, the fraction of CD11chigh DCs
(myeloid DCs) was significantly reduced and the
fraction of CD11cint DCs (plasmacytoid DCs) tended
to be increased (Fig. 3A), indicating a decrease of
conventional DCs or an increase of plasmacytoid
DCs. Similarly, CD11b is expressed not only on macrophages but also on granulocytes (19) and natural
killer cells (20). Macrophages were not different
between the LEH- and RBC-transfused mouse
spleens (Fig. 3B).
A part of the antigen-presenting process involves
the induction of B7 molecules (Fig. 1A), which
includes CD80 and CD86 expression and a proinflammatory cytokine response that helps activate
T cells to counteract against the specific antigen
(15,16). The expression of B7 family proteins was
up-regulated by the administration of LEH (Fig. 4).
Although the human hemoglobin contained in LEH
might have induced xenogeneic immune response in
the recipient mice, it may have been too quick, as the
activation was most prominent 1 day after administration, decreased in 7 days, and became equivalent
to RBC transfusion 4 weeks later (Fig. 4A). Therefore, the early increase in B7 family expression in the
LEH-treated mice may indicate, at least, that LEH
administration was not immunosuppressive as in
RBC transfusion. Subsets of DCs examined 1 day
after administration showed significantly different
patterns of activated antigen (B7) expression
between LEH- and RBC-treated mice (Fig. 4B), suggesting that conventional myeloid DCs might have
recognized LEH as a foreign material.
IL-2 productivity of helper T cells interacting with
DCs from mice infused with LEH was examined to
evaluate their antigen-presenting ability. As conventional enzyme-linked immunosorbent assay for IL-2
determination in culture supernatant may not distinguish IL-2-producing cells from nonproducing cells,
we developed double-transgenic OVA23-3/IL-2-GFP
mice in the current study. This transgenic mouse
strain makes it possible to analyze living cells, which
express GFP under the control of IL-2 promoter and
enhancer after OVA or TSST-1 stimulation, providing a new approach for comparing the degree of early
T-cell activation induced by different APCs. Most
GFP + CD4+ T cells express CD25 simultaneously
after stimulation, suggesting that IL-2 secretion is
correlated with the expression of the activation
marker. The CD25+, GFP- cells may include regulatory T cells, although they might occupy only a part of
the CD25+ cell population. In the current study, DCs
from LEH-treated or RBC-transfused mice did not

ARTIFICIAL O2 CARRIER AND ANTIGEN-PRESENTING ACTIVITY

differ in their ability to support IL-2 production and
CD25 expression levels when measured at any of the
time points, 1 day (Fig. 5), 7 days, and 4 weeks following administration.
Although only a specific antigen (OVA) was tested
in the current study, other antigens may induce a
similar response in the host regardless of the presence or absence of LEH, as the primary antigen
recognition process is the same. Similarly, the immunologic cascade following TSST-1 stimulation is also
common for several pathogens. While the presence of
LEH does not seem to alter the primary immunologic response to third-party antigens, the effect of
repeated administrations of LEH on the secondary
immune response may need to be tested next for
future clinical use.
In conclusion, the administration of 20 mL/kg of
LEH did not interfere with antigen presentation by
DCs that express high levels of costimulatory proteins and are able to activate nave T cells to produce
IL-2 in response to both conventional antigen and
bacterial superantigens as efficiently as DCs from
RBC-transfused mice in vitro. Although IL-2 production and/or T cell activation is one of the earliest
events in the serial immune response, such timing is
relevant when the RES is loaded to the heaviest
metabolic burden and concomitantly exposed to
third-party antigens. Afterwards, a large dose of LEH
administration may possibly induce adverse effects
on the later events, including proliferation and/or
production of other cytokines such as IL-4,
interferon-gamma, and IL-17. Therefore, further
analyses may become necessary to clearly identify
the effect of large-dose LEH administration on each
event of the immune response.
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Artif Organs, Vol. 36, No. 2, 2012