Macrophage activation by polysaccharide biological response
modifier isolated from Aloe vera L. var. chinensis (Haw.) Berg.
C. Liua,1, M.Y.K. Leungb,1, J.C.M. Koona, L.F. Zhuc, Y.Z. Huid,
B. Yud, K.P. Funga,b,⁎
aDepartment of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, People's Republic of China
bInstitute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, People's Republic of China
cSouth China Institute of Botany, Chinese Academy of Sciences, Guangzhou, People's Republic of China
dShanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, People's Republic of China
Received 1 February 2006; received in revised form 25 April 2006; accepted 25 April 2006
A mannose-rich polysaccharide biological response modifier (BRM), derived from Aloe vera L. var. chinensis (Haw.) Berg.,
was demonstrated to be a potent murine B- and T-cell stimulator in our previous study. We here report the stimulatory activity of
PAC-I on murine peritoneal macrophage. The polysaccharide when injected into mice enhanced the migration of macrophages to
the peritoneal cavity. Peritoneal macrophage when treated by PAC-I in vitro had increased expression of MHC-II and FcγR, and
enhanced endocytosis, phagocytosis, nitric oxide production, TNF-α secretion and tumor cell cytotoxicity. The administration of
PAC-I into allogeneic ICR mice stimulated systemic TNF-α production in a dose-dependent manner and prolonged the survival of
tumor-bearing mice. PAC-I is thus a potent stimulator of murine macrophage and the in vitro observed tumoricidal properties of
activated macrophage might account for the in vivo antitumor properties of PAC-I. Our research findings may have therapeutic
implications in tumor immunotherapy.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Polysaccharides; Biological response modifier; Aloe vera; Macrophage
Macrophage, which plays a central role in immune
defence mechanism, is one of the research foci of the
immunology community. The phenotype and function
of macrophage are heterogeneous. The progenitor of
macrophage is pluripotent haematopoietic stem cell in
bone marrow, which passes through the monoblast and
promonocyte state to monocytes. Monocytes, entered
blood circulation as quiescent cells, inhabit different
tissues and further differentiate into tissue resident
macrophages such as Kupffer cells in liver ,
alveolar macrophages in lungs , dendritic cells in
lymphoid tissue , osteoclasts in bone  and
microglia in central nervous system . The role of
macrophage in immunity is versatile. Activated
macrophage can act as accessory cells/antigen present-
ing cells (APCs) and sources of cytokines for the
activation of other immune effector cells [6,7]. As
APCs, antigens are engulfed by phagocytosis or
International Immunopharmacology 6 (2006) 1634–1641
⁎Corresponding author. Department of Biochemistry, The Chinese
University of Hong Kong, Shatin, N.T., Hong Kong SAR, People's
Republic of China. Tel.: +852 2609 6873; fax: +852 2603 7732.
E-mail address: email@example.com (K.P. Fung).
1These authors contributed equally to this work.
1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
endocytosis, and internalized antigens are digested into
fragments and antigen peptides are finally associated
with MHC class II molecule on the cell surface for
antigen presentation . The major cytokines pro-
duced by macrophages for the modulation of other
immune cells are proinflammatory cytokines: IL-1, IL-
6 and TNF-α [9,10]. Activated macrophage can also
be effector cell and engaged in the clearance of foreign
invaders or tumor cells directly. As effector cell,
macrophage exhibits cytotoxicity by phagocytosis,
direct cellular cytotoxicity through cell-to-cell contact,
antibody-dependent cellular cytotoxicity through Fc
receptor (CD16)  and the secretion of cytotoxic
cytokines (IL-1, TNF-α) and reactive oxygen species
(ROI, NO, H2O2) [12–14].
The stimuli for the activation of macrophage can
be cytokine and contact from immune cells like Th1
, and foreign invaders such as microbes. Micro-
bial derivatives like Bacille Calmette-Guerin (BCG)
, unmethylated CpG rich DNA  and cell
surface polysaccharides [18–20] when recognized by
pattern recognition receptors (PRRs) are effective
stimuli for the activation of quiescence macrophages
and other immune cells . These immunostimula-
tory foreign substances and immune cytokines are
named biological response modifiers (BRMs) in
general. Polysaccharide BRMs are not limited to
microbial origin but also botanical origin. According
to the sugar compositions, there are three major
groups of polysaccharide BRMs, which are β-1,3-D-
glucans, α/β-1,4-mannan and highly branched poly-
saccharide of very heterogenous monosaccharide com-
positions. β-1,3-D-Glucans are mainly derived from
cell wall or cytoplasmic reserve of fungus [22–24]. α/
β-1,4-mannan is mainly derived from yeast cell wall
 and freshly layer of Aloe leaves [26,27].
Polysaccharide BRMs of heterogenous sugar compo-
sition are mainly derived from pectic materials of
higher plants [28–31].
The isolation and characterization of the polysac-
charide BRM, PAC-I, was described previously .
PAC-I was purified from Aloe vera L. var. chinensis
(Haw.) Berg., which is a variant of Aloe vera
barbadensis Miller and has wide occurrence in
China. PAC-I has been determined to have mannose
as the major monomeric unit. β-1,4-D-Linked
mannose contributes to the polysaccharide main
skeleton. The molecular weight of PAC-I was
10,000 kDa. PAC-I was demonstrated to exhibit
potent stimulatory effects on B and T lymphocytes.
We here report the stimulatory effect of PAC-I on
2. Materials and methods
2.1. Plant materials and extraction
Aloe vera L. var. chinensis (Haw.) Berg. was cultivated by
South China Institute of Botany, Chinese Academy of
Sciences, Guangzhou, People's Republic of China and was
extracted as described previously . Briefly, fleshy layers of
aloe leaves were blended to viscous solution. Insoluble fibers
were removed by centrifugation (865×g for 10 min). The
supernatant was incubated at 4 °C overnight and then
centrifuged at 5000×g for 20 min to remove insoluble
materials. Hydrophobic materials were removed by XAD-4
resin. Unbound materials, which contained the crude polysac-
charide, was fractionated by membrane with MWCO of
50 kDa. PAC-I was resolved from the membrane-retained
fraction by preparative HPLC. All purified PAC-I was tested
for the present of endotoxin by Limulus ameobocyte lysate
gel-clot assay. The detection limit of the LAL kit was 0.05 EU/
ml, which is close to the declared limit (0.03 EU/ml). Samples
with endotoxin level lower than 0.05 EU/ml were considered
negative. PAC-1 of concentration as high as 10 mg/ml was
tested by the LAL reagent and the result was negative. Only
endotoxin negative preparation was used for subsequent
2.2. Migration of peritoneal macrophages
BALB/c albino mice of 6- to 8-week-old were injected
intraperitoneally (i.p.) with 2 ml of 4% thioglycollate broth
(Sigma Chemical Co., St. Louis, MO, USA) or 0.5 ml of PAC-
I (5 mg/ml). Four days after injection, mice were euthanized
and peritoneal exudate cells were collected by lavage with
10 ml of sterile cold PBS. The exudate cells were pelleted and
resuspended in complete medium at 2×106cells/ml. Cell
suspension of 1 ml was added per well of 24-well plate and
was incubated at 37 °C for 1 h. Nonadherent cells were then
removed by washing with warm PBS. Adherent cells were
typsinized and were counted by haemacytometer.
2.3. Expression of MHC-II and FcγR
Peritoneal exudate cells of thioglycollate-treated mice were
collected as described above. Exudate cells were seeded at
8×105cells/well of 48-well plate. Macrophages were adhered
to plate substratum at 37 °C for 1 h. Nonadherent cells were
removed by washing with warm PBS. Adhered macrophages
were cultured in vitro in the present of 500 μg/ml of PAC-I
polysaccharide for 24 h. PAC-I-treated macrophages were
incubated with 1:500 rat anti-mouse CD16/CD32 monoclonal
antibody (BD Pharmingen, San Diego, CA, USA), or 1:500 rat
anti-mouse IA/IE monoclonal antibody (BD Pharmingen, San
Diego, CA, USA) in PBS with 5% rat serum for 30 min on ice.
The antibody-equilibrated cells were washed three times with
rat serum containing PBS and were subsequently incubated
with 1:500 rat anti-mouse FITC-conjugated antibody (Pierce
Biotechnology Inc., Rockford, IL, USA) for 30 min on ice.
1635C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641
The cells were washed three times before analysis by Cytoron
flow cytometer (Johnson and Johnson, New Brunswick, NJ,
2.4. Enhanced endocytosis
Murine peritoneal exudate cells were plated into 96-well
plate at 4×105cells/well. Adhered macrophages were cultured
with various concentrations of PAC-I for 48 h. After treatment,
horse radish peroxidase (HRP) was added to a final
concentration of 1 mg/ml for 1 h. The macrophages were
washed five times with PBS containing 1% FBS before lysis
by 100 μl of 0.5% Triton-X 100. The enzymatic activity of
HRP was determined by adding o-dianisidine (Sigma
Chemical Co., St. Louis, MO, USA) and H2O2in 0.05 M
phosphate-citrate buffer (pH 6.5) for 10 min. Absorbance of
lysate at 460 nm was measured by spectrophotometer.
2.5. Enhanced phagocytosis of opsonized yeast particles
Murine peritoneal exudate cells were plated into 48-well
plate at 8×105cells/well. Adhered macrophages were cultured
with various concentrations of PAC-I for 72 h before
opsonized phagocytosis. The method of Ortega et al. 
was adopted for the assay of macrophage opsonized
phagocytosis with slight modifications. C. albicans (ATCC
2091) was grown in YM Broth (BD Biosciences, Palo Alto,
California, USA) at 37 °C for 24 h. Cells were pelleted at
400×g and washed twice with PBS. Washed cells were
adjusted to 20×106colony-forming units (CFU)/ml in PBS.
The yeasts were inactivated at 100 °C for 1 h. To label the heat-
inactivated yeasts, 1 μg of 7-amino-actinomycin D (7AAD)
(Sigma Chemical Co., St. Louis, MO, USA) was added to 1 ml
of yeast suspension (20×106CFU/ml). Mixture was homog-
enized by vortexing and was then equilibrated at 4 °C for
20 min in dark. Labeled yeasts were pelleted by centrifugation
(400×g, 10 min). To opsonize the yeasts, 1 ml of heat-
inactivated 7AAD-labeled yeast suspension (20×106CFU/ml)
were incubated with 3 ml of normal mouse serum (Pierce
Biotechnology Inc., Rockford, IL, USA) with shaking at 37 °C
for 30 min. Opsonized yeasts were washed at 400×g and
resuspended in 1 ml of PBS. Peritoneal macrophages were
incubated with stained C. albicans (70 yeasts per peritoneal
cell) at 37 °C for 1 h. After incubation, the cells were washed
three times with PBS (100×g, 4 °C, 10 min) to remove free
yeast particles. Cells were lysed by 1% Nonidet P-40. The
amount of fluorescence in cell lysate was measured by
fluorometer LS-50 (Perkin-Elmer, Norwalk, CT, USA). The
filter settings were 555 nm for excitation and 655 nm for
2.6. Nitric oxide production
Peritoneal exudate cells were plated into 24-well plate at
2×106cells/well. Adhered macrophages were cultured in
MEM (supplemented with 10% fetal bovine serum) with
various concentrations of PAC-I for 48 h. Total nitrate/nitrite
was determined by nitrate/nitrite colorimetric assay kit
(Cayman chemical, Ann Arbor, MI, USA).
2.7. In vitro TNF-α production by peritoneal macrophages
Peritoneal exudate cells were plated into 96-well plate at
4×105cells/well. Adhered macrophages were incubated
with either LPS (5 μg/ml) or various concentrations of PAC-I
at 37 °C for 48 h. After incubation, conditioned medium was
removed for the assay of TNF-α concentration by ELISA kit
(Pierce Biotechnology Inc., Rockford, IL, USA).
2.8. In vitro cytotoxicity
Peritoneal cells were seeded at 1×106cells/well of 96-well
plate. Adhered macrophages were incubated with either LPS
(5 μg/ml) or various concentrations of PAC-I for 24 h. One
million of L-929 cells (target cells) were labeled at 37 °C for
1 h with 100 μCi Na2
MA, USA). Labeled cells were washed and resuspended in
complete medium at 5×104cells/ml. Target cells of 5000
(100 μl) was overlayed on treated peritoneal macrophages.
Cytotoxic reaction was proceeded at 37 °C for 4 h. Supernatant
was then removed for radioactivity counting. The percentage
of specific cytolysis was calculated as follows:
51CrO4(New England Nuclear, Boston,
% specific cytolysis
¼experimental release ðcpmÞ−spontaneous release ðcpmÞ
maximum release ðcpmÞ−spontaneous release ðcpmÞ
2.9. In vivo TNF-α production
Allogeneic ICR mice of 6 to 8 weeks old were injected
intravenously with either 200 μl of LPS (10 μg/ml) or with
various concentrations of PAC-I in endotoxin free sterile PBS.
Blood was collected from PAC-I-treated mice at 1.5 h after
injection by heart puncture. Sera of collected blood samples
were obtained for ELISA.
2.10. Survival test
A group of 10 allogeneic ICR mice of 6 to 8 weeks old
were inoculated i.p. with 1×105Erlich ascites tumor (EAT)
cells. Tumor inoculated mice were injected i.p. with either
PBS, zymosan or PAC-I for 10 consecutive days after
tumor inoculation. Zymosan and PAC-I were administered
at 2 mg/mouse.
3.1. Macrophage migration
The i.p. injection of PAC-I into BALB/c mice attracted
macrophages to reside the peritoneal cavity. Thioglycollate
was used as a positive control. PAC-I injection increased the
1636C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641
number of peritoneal macrophage by at least five folds (Fig. 1).
This demonstrated PAC-I was a chemoattractant to murine
macrophages. However, the effect can be mediated either
directly or indirectly through the secretion of cytokines by
other immune cells, which were stimulated by PAC-1 in
3.2. Expression of MHC-II and Fcγ receptor
The activation of macrophage by PAC-I at molecular level
was studied by monitoring the expression of macrophage
activation markers: MHC-II and Fcγ receptor. Peritoneal
macrophage was incubated with PAC-I and then labeled with
anti-MHC-II or anti-Fcγ receptor antibody, and FITC-
conjugated second antibody for flow cytometry. LPS was the
positive control. The expression of both MHC-II and Fcγ
receptor was up-regulated (Fig. 2).
3.3. Endocytosis and phagocytosis
The internalization of extracellular materials in the form of
endocytosis or phagocytosis is an important indicator of
Fig. 1. Effect of PAC-I on in vivo macrophage migration. Number of
peritoneal macrophages was counted 4 days after i.p. injection of PAC-
I. The number of peritoneal macrophages of PAC-I-treated mice is
significantly higher than that of PBS-treated group. All values
represent mean±S.E.M. of 10 mice.⁎means significantly different
from control at p<0.05.
Fig. 2. Flow cytometric analysis of the expression of MHC-II and Fcγ receptor. Treated peritoneal macrophages were labeled first with rat anti-mouse
CD16/CD32 monoclonal antibody or anti-mouse IA/IE monoclonal antibody, and followed by rat anti-mouse FITC-conjugated antibody. (A)
Fluorescence intensity histogram of PAC-I-treated macrophages labeled with rat anti-mouse CD16/CD32. (B) Fluorescence intensity histogram of
LPS-treated macrophages labeled with rat anti-mouse CD16/CD32. (C) Fluorescence intensity histogram of PAC-I-treated macrophages labeled with
rat anti-mouse IA/IE. (D) Fluorescence intensity histogram of LPS-treated macrophages labeled with rat anti-mouse IA/IE. Open histogram: PBS-
treated macrophages, closed histogram: PAC-I- or LPS-treated macrophages.
1637 C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641
macrophage effector activity. Endocytosis and phagocytosis
are important for the uptake of soluble and particulate antigens
[8,33]. To study endocytosis, HRP was the target for
internalization. The activity of HRP inside macrophages was
proportional to the endocytic activity of the macrophages. The
endocytic activity of PAC-I-treated macrophages was 3.2 folds
more active than the medium control (Table 1).The phagocytic
activity of macrophages was monitored by measuring the
amount of fluorescence-labeled zymosan internalized in
macrophages. The higher the fluorescence, the more active is
the phagocytosis. Similar to endocytosis, the phagocytic
activity of macrophage was enhanced by PAC-I. PAC-I-
treated macrophages were 2.7 folds more active in phagocy-
tosis than medium control (Table 1).
3.4. Nitric oxide production
Nitric oxide is one of the major reactive oxygen species
produced by macrophage for the destruction of targets. Since
the end product of NO is either nitrite or nitrate, the sum of
nitrite and nitrate level provides an indirect measurement of
NO level. When murine macrophages were incubated with
various concentrations of PAC-I, the NO production increased
in a dose-dependent manner. The NO increment plateaued at
about 250 μg/ml of PAC-I (Fig. 3). Zymosan, as a positive
control, also induced NO production in a dose-dependent
3.5. In vitro TNF-α secretion
TNF-α is the tumor cytotoxic cytokine produced by
macrophage. The level of TNF-α secreted by PAC-I-activated
macrophages was monitored by ELISA method and was
compared with control. The levelof TNF-α secreted by PAC-I-
stimulated macrophages was significantly higher than medi-
um-treated macrophages and increased in a dose-dependent
manner. The TNF-α secretion plateaued at 500 μg/ml of PAC-I
(Fig. 4). LPS of 5 μg/ml was the positive control and the level
of TNF-α induced by LPS treatment was 1720±91 pg/ml.
3.6. In vitro tumor cell cytotoxicity
The activation of murine macrophages by PAC-I was
further elucidated in terms of tumor cell cytotoxicity by51Cr
release assay. Mouse connective tissue tumor cell L-929 was
macrophages. The release of
reflected the lysis of L-929 cells by macrophage and was
monitored by gamma counting. The lysis of L-929 cells by
PAC-I-stimulated macrophages was 40% more than untreated
macrophages at 250 μg/ml of PAC-I (Fig. 5). Zymosan was the
positive control and had about 25% more tumor cell lysis than
51Cr and was mixed with PAC-I-stimulated
51Cr into culture medium
Effect of PAC-I on endocytic and phagocytic activity of murine
Endocytosis index Phagocytosis index
PAC-I (500 μg/ml)
LPS (5 μg/ml)
IL-4 (40 ng/ml)
Treated macrophages were lysed by detergent. The amount of HRP in
macrophage lysate of endocytic assay was determined by adding o-
dianisidine and spectrophotometric measurement of the intensity of
colored product. The fluorescence in macrophage lysate of phagocytic
assay was determined by fluorometer. Index was calculated by
dividing reading from treated group by reading of medium-treated
group. All treatments yield index values significantly higher than that
of medium-treated group when analyzed by Student's t-test at p<0.05.
All index values are means±S.E.M. of triplicates. N/A means not
Fig. 3. Nitric oxide production of PAC-I-treated macrophages.
Medium of PAC-I-treated macrophages was collected for total
nitrate/nitrite determination by nitrate/nitrite colorimetric assay kit.
All data represent means±S.E.M. of triplicates.
Fig. 4. TNF-α secretion by PAC-I-treated macrophages in vitro.
Macrophages were cultured with various concentration of PAC-I or
LPS (10 μg/ml) for 2 days. Conditioned medium was removed for
the assay of TNF-α concentration by ELISA kit. All data represent
means±S.E.M. of triplicates.
1638C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641
3.7. In vivo TNF-α secretion and survival assay
In vivo study is the ultimate assay to verify the antitumor
potential of PAC-I. The in vivo tumor cytotoxic effect of PAC-
I was monitored by in vivo TNF-α production and the survival
of tumor-bearing mice. The serum TNF-α level increased in a
dose-dependent manner and displayed a biphasic response
(Fig. 6). Serum TNF-α level (1039 pg/ml) peaked at a dose of
50 μg/mouse of PAC-I and the level declined to 600 pg/ml
when PAC-I dosage increased to 200 μg/mouse. The antitumor
activity of PAC-I has been demonstrated previously .
However, it is not clear whether PAC-I is capable of
prolonging the survival of tumor-bearing mice. Survival
assay demonstrated that PAC-I treatment can prolong the
survival of EAT-bearing mice (Fig. 7). Control group took
about 3 weeks to reach 100% penetrance and 4 weeks for
zymosan (positive control). It took about 6 weeks for PAC-I-
treated group to reach 100% penetrance.
The percentage compositions of mannose, galactose,
glucose and arabinose were 91.5%, 6.0%, 1.0% and
1.5% . No protein was detected in PAC-1 .
Since PAC-1 is composed exclusively of polysaccha-
ride, the receptor of PAC-1 must be lectin. Well-
characterized lectins on macrophage include class A SR,
β-glucan receptor, mannose receptor and complement
receptor type 3. The polysaccharide ligand of Class A
SR is fucoidan . β-Glucan receptor/dectin-1 has
been reported to bind β-1,3-glucan [35–37]. Mannose
receptor binds mannosyl/fucosyl or GlcNAc-glycocon-
jugate ligands . CR3 recognizes ligands such as
intercellular adhesion molecule-1 (ICAM-1), fixed iC3b
and β-glucan . Since there is only 1% of glucose in
PAC-1, it is very unlikely that PAC-1 activates
macrophage through β-glucan receptor or CR3 but
mannose receptor. Therefore, it is reasonable to
conceive the receptor of PAC-I in vivo is mannose
The binding of PAC-I to mannose receptor of
quiescence macrophages triggered cellular events and
activated the macrophages. PAC-I-activated macro-
phages displayed up-regulation of MHC-II molecules
Fig. 5. Cytotoxic activity of PAC-I-treated macrophages in vitro.
Macrophages were cultured with various concentration of PAC-I or
LPS (10 μg/ml) for 1 day. Treated macrophages were mixed with51Cr-
labeled L-929cells (target cells) for 4 h. Culturedmediumwas counted
for gamma radioactivity. Cell lysis was expressed as percentage of
specific cytolysis. The equation for the calculation was described in
Section 2. All data represent means±S.E.M. of triplicates.⁎means
significantly different from control at p<0.05.
Fig. 6. Systemic TNF-α secretion by PAC-I-injected mice. Group of
five allogeneic ICR mice were injected i.v. with either PBS,LPS (2 μg/
mouse) or various dosages of PAC-I in endotoxin-free sterile PBS.
Sera were collected at 1.5 h after injection for the determination of
TNF-α level by ELISA kit. All data represent means±S.E.M. of five
Fig. 7. Survival of PAC-I-injected EAT-bearing mice. Group of 10 ICR
mice were inoculated i.p. with 1×105EAT cells. Tumor-inoculated
mice were injected i.p. with either PBS, zymosan (2 mg/mouse/day) or
PAC-I (2 mg/mouse/day) for 10 consecutive days after tumor
1639C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641
and Fcγ receptor. The enhancement of endocytosis and
phagocytosis by PAC-I imposed the activated macro-
phages greater capability to uptake soluble and
particulate antigens for processing, and the increased
expression of MHC-II enabled the activated macro-
phages greater antigen presenting capacity. The in-
creased expression of Fcγ receptor conceivably
rendered the activated macrophages higher capacity to
clear antibody–antigen or antibody–tumor cell com-
plex. PAC-I also augmented the killing mechanisms of
macrophages, which included phagocytosis, the pro-
duction of toxic NO and the secretion of TNF-α. The
enhanced effector function of PAC-I-activated macro-
phages was demonstrated by increased tumor cell
cytotoxicity in vitro. The in vivo antitumor action of
PAC-I in the murine model studied was likely a
consequence of the direct activation of macrophage by
PAC-I. There are evidences suggesting macrophage,
which is a key player in immunosurveillance process, in
the initiation of specific antitumor immune response and
in tumor clearance, is suppressed by tumor cells .
The activation of macrophages by BRM-like PAC-I
may restore or even enhance the antigen recognition,
antigen presentation and tumor killing capacity of
dysfunction macrophage. Based on this notion, macro-
phage is an excellent immunopotentiation target for
The successful cure of various cancers by modern
medical treatments is still limited. In most circum-
stances, the treatments only prolonged the survival of
patients rather than radical treatments. The use of
complementary and alternative medicine (CAM) or
phytomedicine as an agent to improve the quality of life
of cancer patients or as an alternative for cancer therapy
is raising the concern of public, scientists and healthcare
professionals [41–48]. As a polysaccharide BRM, PAC-
I is rich in Aloe vera L. var. chinensis (Haw.) Berg. and
can be purified economically by simple purification
procedures, is a potential phytomedicine for tumor
In conclusion, PAC-I is a potent murine macrophage
stimulator. PAC-I-activated macrophages have elevated
expression of macrophage activation markers and have
enhanced effector cell functions, and tumor cell
cytotoxic activity. PAC-I when administered in vivo is
capable of prolonging the survival of tumor-bearing
5. Statistical analysis
Student's t-test was used for all the statistical analysis
of data at p<0.05.
This project was partially funded by Earmarked grant
from Research Grant Council of Hong Kong SAR
 Naito M, Hasegawa G, Takahashi K. Development, differenti-
ation, and maturation of Kupffer cells. Microsc Res Tech
 Naito M. Macrophage heterogeneity in development and
differentiation. Arch Histol Cytol 1993;56:331–51.
 Wu L, Vandenabeele S, Georgopoulos K. Derivation of dendritic
cells from myeloid and lymphoid precursors. Int Rev Immunol
 Roodman GD. Cell biology of the osteoclast. Exp Hematol 1999;
 Cuadros MA, Navascues J. The origin and differentiation of
microglial cells during development. Prog Neurobiol 1998;
 Unanue ER. Perspective on antigen processing and presentation.
Immunol Rev 2002;185:86–102.
 Murtaugh MP, Foss DL. Inflammatory cytokines and antigen
presenting cell activation. Vet Immunol Immunophathol 2002;
 Neild A, Murata T, Roy CR. Processing and major histocom-
patibility complex class II presentation of Legionella pneumo-
phila antigens by infected macrophages. Infect Immun 2005;
 Giacomini E, Iona E, Ferroni L, Miettinen M, Fattorini L, Orefici
G, et al. Infection of human macrophages and dendritic cells with
Mycobacterium tuberculosis induces a differential cytokine gene
expression that modulates T cell response. J Immunol 2001;
 KelkP,ClaessonR, HanstromL,Lerner UH,KalfasS, Johansson
A. Abundant secretion of bioactive interleukin-1beta by human
macrophages induced by Actinobacillus actinomycetemcomitans
leukotoxin. Infect Immun 2005;73:453–8.
 Charak BS, Agah R, Mazumder A. Granulocyte–macrophage
colony-stimulating factor-induced antibody-dependent cellular
cytotoxicity in bone marrow macrophages: application in bone
marrow transplantation. Blood 1993;81:3474–9.
 Haidaris CG, Bonventre PF. A role for oxygen-dependent
mechanisms in killing of Leishmania donovani tissue forms by
activated macrophages. J Immunol 1982;129:850–5.
 Mateo RB, Reichner JS, Albina JE. NO is not sufficient to
explain maximal cytotoxicity of tumoricidal macrophages
againstan NO-sensitive cell line. JLeukocBiol 1996;60:245–52.
 Remer KA, Reimer T, Brcic M, Jungi TW. Evidence for
involvement of peptidoglycan in the triggering of an oxidative
burst by Listeria monocytogenes in phagocytes. Clin Exp
 Rilsgaard S, Rhodes JM, Bennedsen J. Macrophage activation by
lymphokines and after direct contact with sensitized lympho-
cytes: histocompatibility requirements and the effect of inhibi-
tors. Scand J Immunol 1978;7:209–13.
 Juy D, Chedid L. Comparison between macrophage activation
and enhancement of nonspecific resistance to tumors by
mycobacterial immunoadjuvants. Proc Natl Acad Sci U S A
1640C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641
 Krieg AM. Mechanisms and applications of immune stimulatory Download full-text
CpG oligodeoxynucleotides. Biochim Biophys Acta 1999;
 Sironi M, Sica A, Riganti F, Licciardello L, Colotta F, Mantovani
A. Interleukin-6 gene expression and production induced in
human monocytes by membrane proteoglycans from Klebsiella
pneumoniae. Int J Immunopharmacol 1990;12:397–402.
 Blackstock R, McElwee N, Neller E, Shaddix-White J.
Regulation of cytokine expression in mice immunized with
cryptococcal polysaccharide, a glucuronoxylomannan (GXM),
associated with peritoneal antigen-presenting cells APC):
requirements for GXM, APC activation, and interleukin-12.
Infect Immun 2000;68:5146–53.
 Heinzelmann M, Polk Jr HC, Chernobelsky A, Stites TP, Gordon
LE. Endotoxin and muramyl dipeptide modulate surface receptor
expression on human mononuclear cells. Immunopharmacology
 Gordon S. Pattern recognition receptors: doubling up for the
innate immune response. Cell 2002;111:927–30.
 Domer J, Elkins K, Ennist D, Baker P. Modulation of immune
responses by surface polysaccharides of Candida albicans. Rev
Infect Dis 1988;10(Suppl 2):S419–22.
 Blaschek W, Kasbauer J, Kraus J, Franz G. Pythium aphani-
dermatum: culture, cell-wall composition, and isolation and
structure of antitumour storage and solubilised cell-wall (1–3),
(1–6)-beta-D-glucans. Carbohydr Res 1992;231:293–307.
 Leung MY, Fung KP, Choy YM. The isolation and characteriza-
tion of an immunomodulatory and anti-tumor polysaccharide
preparation from Flammulina velutipes. Immunopharmacology
 Garner RE, Hudson JA. Intravenous injection of Candida-
derived mannan results in elevated tumor necrosis factor alpha
levels in serum. Infect Immun 1996;64:4561–6.
 Leung MY, Liu C, Zhu LF, Hui YZ, Yu B, Fung KP. Chemical
and biological characterization of a polysaccharide biological
response modifier from Aloe vera L. var. chinensis (Haw.) Berg.
 Ni Y, Turner D, Yates KM, Tizard I. Isolation and characteriza-
tion of structural components of Aloe vera L. leaf pulp. Int
 Nose M, Terawaki K, Oguri K, Ogihara Y, Yoshimatsu K,
Shimomura K. Activation of macrophages by crude polysaccha-
ride fractions obtained from shoots of Glycyrrhiza glabra and
hairy roots of Glycyrrhiza uralensis in vitro. Biol Pharm Bull
 Guo Y, Matsumoto T, Kikuchi Y, Ikejima T, Wang B, Yamada H.
Effects of a pectic polysaccharide from a medicinal herb, the
roots of Bupleurum falcatum L. on interleukin 6 production of
murine B cells and B cell lines. Immunopharmacology 2000;
 Yaneva MP, Botushanova AD, Grigorov LA, Kokov JL,
Todorova EP, Krachanova MG. Evaluation of the immunomod-
ulatory activity of Aronia in combination with apple pectin in
patients with breast cancer undergoing postoperative radiation
therapy. Folia Med 2002;44:22–5.
 Ebringerova A, Kardosová A, Hromádková Z, Hríbalová V.
Mitogenic and comitogenic activities of polysaccharides from
some European herbaceous plants. Fitoterapia 2003;74:52–61.
 Ortega E, Algarra I, Serrano MJ, Alvarez de Cienfuegos G,
Gaforio JJ. The use of 7-amino-actinomycin D in the analysis of
Candida albicans phagocytosis and opsonization. J Immunol
 Norbury C, Hewlett L, Prescott A, Shastri N, Watts C. Class I
MHC presentation of exogenous soluble antigen via macro-
pinocytosis in bone marrow macrophages. Immunity 1995;3:
 Kim WS, Ordija CM, et al. Activation of signaling pathways by
putative scavenger receptor class A (SR-A) ligands requires
CD14 but not SR-A. Biochem Biophys Res Commun 2003;
 Czop JK, Austen KF. A beta-glucan inhibitable receptor on
human monocytes: its identity with the phagocytic receptor for
particulate activators of the alternative complement pathway.
J Immunol 1985;134:2588–93.
 Czop JK, Kay J. Isolation and characterization of beta-glucan
receptors on human mononuclear phagocytes. J Exp Med
 Brown GD, Taylor PR, et al. Dectin-1 is a major beta-glucan
receptor on macrophages. J Exp Med 2002;196:407–12.
 Zamze S, Martinez-Pomares L, et al. Recognition of bacterial
capsular polysaccharides and lipopolysaccharides by the macro-
phage mannose receptor. J Biol Chem 2002;277:41613–23.
 Ross GD, Vetvicka V. CR3 (CD11b, CD18): a phagocyte and NK
cell membrane receptor with multiple ligand specificities and
functions. Clin Exp Immunol 1993;92:181–4.
 Foss FM. Immunologic mechanisms of antitumor activity. Semin
 Cohen I, Tagliaferri M, Tripathy D. Traditional Chinese medicine
in the treatment of breast cancer. Semin Oncol 2002;29:563–74.
 Jatoi A, Ellison N, Burch PA, Sloan JA, Dakhil SR, NovotnyP, et
al. A phase II trial of green tea in the treatment of patients with
androgen independent metastatic prostate carcinoma. Cancer
 Nahin RL. Use of the best case series to evaluate complementary
and alternative therapies for cancer: a systematic review. Semin
 Oh WK, Small EJ. Complementary and alternative therapies in
prostate cancer. Semin Oncol 2002;29:575–84.
 Pongnikorn S, Fongmoon D, Kasinrerk W, Limtrakul PN. Effect
of bitter melon (Momordica charantia Linn) on level and
function of natural killer cells in cervical cancer patients with
radiotherapy. J Med Assoc Thai 2003;86:61–8.
 Shen J, Andersen R, Albert PS, Wenger N, Glaspy J, Cole M, et
al. Use of complementary/alternative therapies by women with
advanced-stage breast cancer. BMC Complement Altern Med
 White JD. Complementary and alternative medicine research: a
National Cancer Institute perspective. Semin Oncol 2002;
 Yoshimura K, Ichioka K, Terada N, Terai A, Arai Y. Use of
complementary and alternative medicine by patients with
localized prostate carcinoma: study at a single institute in
Japan. Int J Clin Oncol 2003;8:26–30.
1641 C. Liu et al. / International Immunopharmacology 6 (2006) 1634–1641