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IMMUNE NETWORK http://www.ksimm.o r.kr Vol ume 9 Number 6 December 2009
DOI 10.4110/in.2009.9.6.255
PISSN 1598-2629
255
ORIGINAL ARTICLE
Received on December 7, 2009. Revised on December 14, 2009. Accepted on December 16, 2009.
CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial
License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribu-
tion, and reproduction in any medium, provided the original work is properly cited.
*Corresponding Author. Tel: 82-2-3399-1601; Fax: 82-2-3399-1617; E-mail: kimkj@syu.ac.kr
Keywords: Cordycepin, Adenosine, M1/M2 phenotype, Morphology change
Role of Cordycepin and Adenosine on the Phenotypic Switch
of Macrophages via Induced Anti-inflammatory Cytokines
Seulmee Shin1,3, Sunhee Moon1, Yoonhee Park1, Jeonghak Kwon1, Seungjeong Lee2, Chong-Kil Lee2, Kyunghae Cho3,
Nam-Joo Ha1 and Kyungjae Kim1*
College of Pharmacy, 1Sahmyook University, Seoul 139-742, 2Chungbuk University, Cheongju 361-763, 3Department of Biology, Seoul
Women’s University, Seoul 139-774, Korea
Background: Chronic low grade inflammation is closely
linked to type II diabetes, obesity, and atherosclerosis.
Macrophages play a key role in the regulation of pro- or an-
ti-inflammatory actions at the lesion sites of disease.
Components of cordyceps militaris, cordycepin and ad-
enosine, have been used for the modulation of inflammatory
diseases. The effects of cordycepin in the modulation of mac-
rophages have yet to be elucidated. We investigated the ef-
fects of cordycepin and adenosine on the morphological
changes of macrophages under the inflammatory condition
of LPS and an anti-inflammatory condition involving high
concentrations of adenosine. Methods: We c o n f irmed the
mRNA levels of the M1/M2 cytokine genes through RT-PCR
and morphological change. Results: LPS-activated macro-
phages returned to their inactivated original shape, i.e., they
looked like naïve macrophages, through the treatment with
high concentrations of cordycepin (40 μg/ml). LPS and ad-
enosine activated macrophages also returned to their origi-
nal inactivated shapes after cordycepin treatment; however,
at relatively higher levels of cordycepin than adenosine. This
change did not occur with relatively low concentrations of
cordycepin. Adenosine down-regulated the gene expression
of M1 cytokines (IL-1β, TNF-α) and chemokines (CX3CR1,
RANTES), as well as cordycepin. Additionally, M2 cytokines
(IL-10, IL-1ra, TGF-β) were up-regulated by both cordycepin
and adenosine. Conclusion: Based on these observations,
both cordycepin and adenosine regulated the phenotypic
switch on macrophages and suggested that cordycepin and
adenosine may potentially be used as immunomodulatory
agents in the treatment of inflammatory disease.
[Immune Network 2009;9(6):255-264]
INTRODUCTION
Most arthritis, diabetes, and heart disease sufferers are all too
familiar with inflammation. Inflammation is the activation of
the immune system in response to infection, irritation, or in-
jury and is characterized by an influx of white blood cells,
redness, heat, swelling, pain, and dysfunction of the organs
involved (1,2). Macrophages play a key role in the immune
system via the regulation of pro-inflammatory and anti-in-
flammatory actions at the lesion sites of disease. In injured
tissues, monocytes/macrophages infiltrate inflamed tissues
and play central roles (3). Mediators of theses event including
pro-inflammatory cytokines and chemokines promote macro-
phage accumulation via increasing the expression of adhesion
molecules (3,4). In these states, macrophages are called clas-
sically activated M1 macrophages (M1), and are induced by
IFN-
γ
either alone or in concert with microbial stimuli (e.g.
lipopolysaccharides (LPS)) or cytokines (e.g. TNF-
α
and
GM-CSF) (5). Anti-inflammatory cytokines such as IL-4, IL-10,
and IL-13 have been found to be more than simple inhibitors
of macrophage activation, but also able to induce an alter-
native M2 form of macrophage activation (M2) (6).
Taken together, macrophage phenotype is an important
factor in recruiting macrophages to the lesion site, scavenging
pathogens, expressing surface proteins, and releasing pro-
and anti-inflammatory cytokines.
Cordycepin, 3
’
-deoxyadenosine, is an effective component
isolated from
Cordyceps militaris
. Cordycepin has various bi-
Role of Cordycepin and Adenosine on the Phenotypic Switch of Macrophages
Seulmee Shin, et al.
256 IMMUNE NETWORK http://www. ksimm.or.kr Volume 9 Number 6 December 2009
ological effects including anti-fungal (7), anti-malarial (8), an-
ti-herpes (9), anti-tumorigenic on some cell lines, anti-leuke-
mic activities (10), and anti-diabetic effects (11).
Adenosine is an endogenous purine nucleoside that, fol-
lowing its release from cells or after being formed ex-
tracellularly, diffuses to the cell membrane of surrounding
cells where it binds specific cell-surface structures that recog-
nize it, termed adenosine receptors (12,13). The role of ad-
enosine as an extracellular signaling molecule demonstrated
that adenosine is both a potent negative inotropic agent and
a coronary vasodilator (14).
Taken together, cordycepin and adenosine must represent
excellent immunomodulators regulating immune cell activa-
tion via the suppression of over-expressed inflammation.
However, the effects of neither cordycepin nor adenosine on
morphological change in macrophages have been determi-
ned.
In the current study, we investigated the effects of cordyce-
pin and adenosine on morphological changes in macrophage
under various experimental conditions.
MATERIALS AND METHODS
Reagents
Cordycepin, adenosine, lipopolysaccharide (LPS), and 3-(4,5-
dimethylthiazol-2-yl)2,5-dephenyltetrazolium bromide (MTT)
were purchased from Sigma (St. Louis, MS, USA). Dulbecco
’
s
Modified Eagle Medium (DMEM), antibiotic-penicillin/strepto-
mycin solution, and fetal bovine serum (Hyclone, Logan, UT,
USA) were used for cell culture.
Cell culture
RAW 264.7 cells, a murine macrophage cell line, was main-
tained in a 95% air, 5% CO
2
atmosphere in DMEM supple-
mented with 10% heat-inactivated fetal bovine serum (FBS),
10,000 U/ml penicillin, and 10,000 U/ml streptomycin.
Cytotoxicity assay
Cell viability was determined by a MTT reduction assay. In
brief, RAW 264.7 cells were pre-incubated overnight in
24-well plates at a density of 2×10
5
cells per well, and were
then washed with 1x PBS. Cells were then treated with differ-
ent concentrations of cordycepin or adenosine for 24 hrs and
grown in 0.5 mg/ml MTT at 37
o
C for 4 hrs. After removing
culture supernatants, the resulting dark blue crystals were dis-
solved with DMSO (Dimethyl sulfoxide). Absorbance values
were read at 560 nm using an automated VERSAmax micro-
plate reader (Molecular Devices, Sunnyvale, CA, USA). All de-
terminations were confirmed by replication in at least three
independent experiments.
Reverse transcription (RT)-PCR analysis
Total RNA was prepared from cells using the RNeasy Mini
kit (QIAGEN, Valencia, CA, USA) according to the protocol
for the manufacturer. For RT-PCR, 1
μ
g/ml of total RNA from
each sample was reverse transcribed for 1 hr at 37
o
C in a
reaction mixture containing 2 mM deoxynucleotide triphos-
phate (dNTP), 2
μ
M oligo-dT primer, 1x reverse transcriptase
buffer, 200 units MMLV reverse transcriptase, and 1 U RNase
inhibitor (invitrogen, Carlsbad, CA, USA). Next, PCR was per-
formed using the above-prepared cDNA as a template, with
the following cycle parameters: 94
o
C, 2 min; 35 cycles, 94
o
C,
45 s; 62
o
C, 45 s, 72
o
C, 1 min; 72
o
C, 5 min, 4
o
C,
∞
. All PCR
products were visualized by electrophoresis in 1.5% agarose
gels, followed by straining with ethidium bromide (EtBr; 0.5
μ
g/ml).Verification of specific genes was established using
their predicted sizes under ultraviolet (UV) light. The primer
sequences were as follows: 5'-CAG GAT GAG GAC ATG ACA
CC-3' (forward), 5'-CTC TGC AGA CTC AAA CTC CAC-3'
(reverse) for IL-1
β
; 5'-TGC TGG GTA CTT ACA AGG ACC-3'
(forward), 5'-CAG AGC GGA TGA AGG TAA AGC-3' (reverse)
for IL-1RA; 5'-TGG CCA CAC TTG AGA GCT GC-3' (forward),
5'-TTC AGG GAT GAA GCG GCT GG-3' (reverse) for IL-10;
5'-TTG ACC TCA GCG CTG AGT TA-3' (forward), 5'-CCT
GTA GCC CAC GTC GTA GC-3' (reverse) for TNF-
α
; 5'-ATC
ATC CTC ACT GCA GCC GC-3' (forward), 5'-CAC ACT TGG
CGG TTC CTT CG-3' (reverse) for RANTES; 5'-TCC TGT CCG
TCT TCT ACG CC-3' (forward), 5'-ATT GTG GAG GCC CTC
ATG GC-3' (reverse) for CX3CR1; 5'-CTA AAC CAG GAG TCC
TGC GG-3' (forward), 5'-CTA GTG GCC AAG GTG ATC CC-3'
(reverse) for CX3CL1; 5'-GGA GCT GGT GAA ACG GAA
GC-3' (forward), 5'-CAT AGT AGT CCG CTT CGG GC-3'
(reverse) for TGF-
β
; and 5'-GTG GGC CGC CCT AGG ACC
AG-3' (forward), 5'-GGA GGA AGA GGA TGC GGC AG T-3'
(reverse) for
β
-actin as an internal PCR control. Band in-
tensity was quantified by densitometric analysis (Infinity 3026,
Vilber Lourmat, Marne la Valled, France).
Morphology
To determine the effects of cordycepin or adenosine on mac-
rophage morphology, cells were cultured in 24-well plates at
a density of 2×10
5
cells per well, sterile for 24 hrs. The cul-
Role of Cordycepin and Adenosi ne on the Phenotypic Swit ch of Macrophages
Seulmee Shin, et al.
257IMMUNE NETWORK http://www.ksimm.o r.kr Vol ume 9 Number 6 December 2009
ture medium was then removed, and the cells were treated
with (A) cordycepin (5, 10, 20, 40
μ
g/ml) in the presence
of LPS (100 ng/ml), and (B) adenosine (5, 10, 20, 40
μ
g/ml)
in the absence of LPS, for 24 hrs. Then, to investigate which
combination of cordycepin and adenosine affected the phe-
notypic changes in macrophages, cells were treated with (C)
adenosine (2.5
μ
g/ml)/cordycepin (5
μ
g/ml), adenosine (5
μ
g/ml)/cordycepin (10
μ
g/ml), adenosine (10
μ
g/ml)/cordy-
cepin (20
μ
g/ml), and adenosine (20
μ
g/ml)/cordycepin (40
μ
g/ml), (D) adenosine (20
μ
g/ml)/cordycepin (10
μ
g/ml),
adenosine (40
μ
g/ml)/cordycepin (20
μ
g/ml), and adenosine
(80
μ
g/ml)/cordycepin (40
μ
g/ml), for 24 hrs. Following the
treatment, the culture supernatant was removed and cells
were then fixed and stained in Diff-quick Solution (Baxter,
Houston, TX, USA). Also, to determined the effects of cordy-
cepin on lipid accumulation, cells were cultured in 24-well
plates at a density of 2×10
5
cells per well, sterile for 24 hrs.
The culture medium was removed, and the cells were treated
with cordycepin (5, 10, 20, 40
μ
g/ml) in the presence of LPS
(100 ng/ml) for 24 hrs. Following the treatment, the culture
supernatant was removed and cells were then fixed and
stained in Oil red O solution.
Statistical analysis
Data are expressed as mean±standard deviation. Statistical
significance between the groups was determined by paired
t-test and one-way ANOVA for repeated measures. Results
with p
＜
0.05 were considered statistically significant. Data
were assessed using SPSS (Version 15.0, SPSS Inc., Chicago,
IL, USA).
RESULTS
Effect of cordycepin and adenosine on cell viability
To rule out cordycepin toxicity, we tested its effect on the
viability of RAW 264.7 by MTT assay. Exposure of cells to
cordycepin (5
∼
40
μ
g/ml) or adenosine (2.5
∼
40
μ
g/ml) for
24 hrs showed no significant toxic adverse effects on viability
compare to the untreated controls (Supplementary Fig. 1).
Cordycepin- and adenosine-induced morphologi-
cal changes of macrophages
Morphological examination under a light microscope in-
dicated that more than half of the macrophages appeared as-
teroid and there were little or no phagocytic granules prior
to treatment with cordycepin or ademosine. After being treat-
ed with cordycepin (5
∼
40
μ
g/ml) in the presence of LPS
(100 ng/ml), macrophages in the control group became
round and the granules increased (Fig. 1A) whereas those
without adenosine and LPS appeared larger and rougher than
control (Fig. 1B). These results suggested that cells treated
with high concentration of adenosine (40
μ
g/ml) were similar
to those exposed to LPS. However, cells treated with high
concentrations of cordycepin (40
μ
g/ml) in the presence with
LPS were smoother than those treated with LPS.
Effects of cordycepin and adenosine on the gene
expression of M1 cytokines and chemokines
To further investigate to important role of cordycepin and ad-
enosine on inflammation, murine macrophage cells with cor-
dycepin (5
∼
40
μ
g/ml) in the presence of LPS (100 ng/ml),
or adenosine (10
μ
g/ml) in the absence with LPS, for 24 hrs
exhibited altered M1 cytokines and chemokines. Cordycepin
suppressed IL-1
β
, TNF-
α
, CX3CR1, CX3CL1, and RANTES
expression in a dose-dependent manner. Adenosine exhibited
a slight increase in these genes, while the cells co-treated
with adenosine and LPS decreased their expression (Fig. 2).
Effects of cordycepin and adenosine on the gene
expression of M2 cytokines
We determined M2 cytokines expression on inflammation in
murine macrophage cells with cordycepin (5
∼
40
μ
g/ml) in
the presence of LPS (100 ng/ml) or adenosine (10
μ
g/ml) in
the absence with LPS, for 24 hrs. Cordycepin upregulated
IL-10 and TGF-
β
expression in a dose-dependent manner.
Adenosine increased the expression of these genes in a man-
ner similar to that of LPS (Fig. 3).
Phenotypic switch by cordycepin with adenosine or
adenosine with cordycepin
When cultured in medium alone, normal RAW 264.7 cells
tended to be round, with none appearing to spread over the
surface (Fig. 4). In cells treated with double the concentration
of cordycepin than adenosine, a na
ï
ve macrophage form from
the M1 phenotype was induced (Fig. 4A). On the other hand,
in cells treated with double the concentration of adenosine
than cordycepin, an M2 phenotype from an M1 phenotype
was induced (Fig. 4B).
Lipid accumulation in macrophages
When cultured in medium alone, normal RAW 264.7 cells tend-
ed to be round, with none appearing to spread over the
Role of Cordycepin and Adenosine on the Phenotypic Switch of Macrophages
Seulmee Shin, et al.
258 IMMUNE NETWORK http://www. ksimm.or.kr Volume 9 Number 6 December 2009
Figure 1. RAW 264.7 cells were cultured on cover slips in the presence of various concentrations of cordycepin (A) medium alone (a), LPS
100 ng/ml (b), cordycepin (5μg/ml) (c), 10μg/ml (d), 20μg/ml (e), and 40μg/ml (f), in the presence of LPS and adenosine (B) medium alone
(a), LPS 100 ng/ml (b), adenosine (5μg/ml) (c), 10μg/ml (d), 20μg/ml (e), 40μg/ml (f), and in the absence of LPS for 24 hrs. The cells were
then fixed and stained in Diff-quick Solution (×400).
surface. The cells treated with a high concentration of cordyce-
pin exhibited an increased accumulation of red-stained lipids
as compared to those treated with low concentrations (Fig. 5).
DISCUSSION
The data presented in this paper indicates that cordycepin
Role of Cordycepin and Adenosi ne on the Phenotypic Swit ch of Macrophages
Seulmee Shin, et al.
259IMMUNE NETWORK http://www.ksimm.o r.kr Vol ume 9 Number 6 December 2009
Figure 2. Effects of cordycepin and adenosine on the expression of M1 cytokines (A, B), chemokines, and their receptor (C, D) in RAW 264.7
cells. Cells were incubated in various concentrations of cordycepin (5, 10, 20, 40μg/ml) in the presence of LPS (100 ng/ml) or adenosine (10μg/ml),
in the presence or absence of LPS, for 24 hrs. mRNA levels of M1 cytokine and chemokine genes were determined by RT-PCR analysis. β-actin
was used as a control. ††p＜0.01 vs. cells only; *p＜0.05, **p＜0.01 vs. LPS only; and †p＜0.05 vs. cells only were all based on Student’s
t-tests.
and adenosine regulate immune responses. The present study
demonstrated that cordycepin and adenosine changed the
macrophage phenotypic switch via a decrease in the ex-
pression of pro-inflammatory cytokines and chemokines (IL-1
β
, IL-6, TNF-
α
, RANTES, CX3CL1), and an induction of an-
ti-inflammatory cytokine (IL-1ra, IL-10, TGF-
β
) in RAW 264.7
cells.
In inflamed tissue or lesions, na
ï
ve macrophages are acti-
vated either classically or alternatively depending on the pro-
file of the environmental stimuli. Classically activated macro-
phages (M1), induced by IFN-
γ
plus TNF-
α
or TLRs ligands,
are typical effectors of cell-mediated immunity. Alternative ac -
tivation of macrophages by IL-4 and IL-13 or by phagocytosis
of apoptotic cells antagonizes classical activation. Alternatively
activated macrophages (M2) secrete the anti-inflammatory cy-
tokines IL-1ra, IL-10, and TGF-
β
, thereby providing signals
to deactivate macrophages as well as to mediate im-
munosuppressive and healing processes. The phenotypes of
both M1 and M2 cells in inflamed tissues is the major source
of cytokines, and it is believed that the coordinated switching
between these two phenotypes determines the outcome of
inflammatory processes (15). These cytokines affect wound
healing rate and wound-breaking strength while M1 cells pro-
duce pro-inflammatory cytokines and chemokines, IL-1
β
,
IL-6, TNF-
α
, RANTES, and CX3CL1, and attract more atten-
tion in that they can be localized to the infected tissue, man-
ifested systemically throughout the body, and cause vaso-
dilation as well as symptoms of inflammation, such as red-
ness, swelling, heat, and pain (16). Cordycepin down-regu-
lated the expression of pro-inflammatory molecules such as
Role of Cordycepin and Adenosine on the Phenotypic Switch of Macrophages
Seulmee Shin, et al.
260 IMMUNE NETWORK http://www. ksimm.or.kr Volume 9 Number 6 December 2009
Figure 2. Continued.
IL-1
β
and TNF-
α
, and the chemokines, CX3CR1 and
RANTES, in LPS-stimulated RAW 264.7 cells (Fig. 2). When
we examined the morphological changes that took place in
macrophages treated with cordycepin and LPS, cells treated
with LPS and low concentrations of cordycepin (5
∼
10
μ
g/ml)
were similar those exposed to LPS alone. However, cells
treated with high concentrations of cordycepin (40
μ
g/ml) in
combination with LPS were smoother than those treated with
LPS alone (Fig. 1A). Also, these levels inhibited the activation
of pro-inflammatory cytokines and related proteins in both of
the LPS-activated cell types (17,18).
Adenosine treated with LPS down-regulated the expression
Role of Cordycepin and Adenosi ne on the Phenotypic Swit ch of Macrophages
Seulmee Shin, et al.
261IMMUNE NETWORK http://www.ksimm.o r.kr Vol ume 9 Number 6 December 2009
Figure 3. Effects of cordycepin (A) and adenosine (B) on the expression of M2 cytokines in RAW 264.7 cells. Cells were incubated in various
concentrations of cordycepin (5, 10, 20, 40μg/ml) in the presence of LPS (100 ng/ml), or adenosine (10μg/ml), in the presence or absence
of LPS, for 24 hrs. mRNA levels of M2 cytokine genes were determined by RT-PCR analysis. β-actin was used as a control. ††p＜0.01 vs.
cells only; *p＜0.05, **p＜0.01 vs. LPS only; and †p＜0.01 vs. cells only were based on Student’s t-tests.
of pro-inflammatory cytokines and chemokines; however,
when we exposed them to adenosine, it induced gene ex-
pression in macrophages somewhat (Fig. 2). Additionally,
with respect to morphological change, low concentrations of
adenosine activated macrophages and high concentration of
adenosine induced M2 cells that contained lipid vacuoles
Role of Cordycepin and Adenosine on the Phenotypic Switch of Macrophages
Seulmee Shin, et al.
262 IMMUNE NETWORK http://www. ksimm.or.kr Volume 9 Number 6 December 2009
Figure 4. The morphological switch by cordycepin with adenosine or adenosine and cordycepin. RAW 264.7 cells were cultured on cover slip
s
in the presence of double the concentration of cordycepin than adenosine (A) in: medium alone (a), LPS 100 ng/ml (b), adenosine (2.5μg/ml)
/
cordycepin (5μg/ml) (c), adenosine (5μg/ml)/cordycepin (10μg/ml) (d), adenosine (10μg/ml)/cordycepin (20μg/ml) (e), adenosine (20μg/ml)
/
cordycepin (40μg/ml) (f), and 2x adenosine/cordycepin (B) in: medium alone (a), LPS 100 ng/ml (b), adenosine (20μg/ml)/cordycepin (10μg/ml)
(c), adenosine (40μg/ml)/cordycepin (20μg/ml) (d), adenosine (80μg/ml)/cordycepin (40μg/ml) (e), for 24 hrs. The cells were then fixed and
stained in Diff-quick Solution (×400).
Role of Cordycepin and Adenosi ne on the Phenotypic Swit ch of Macrophages
Seulmee Shin, et al.
263IMMUNE NETWORK http://www.ksimm.o r.kr Vol ume 9 Number 6 December 2009
Figure 5. Lipid accumulation of macrophages by cordycepin. RAW 264.7 cells were cultured on cover slips in the presence of various
concentrations of cordycepin: medium alone (A), LPS (100 ng/ml) (B), cordycepin (5μg/ml) (C), 10μg/ml (D), 20μg/ml (E), 40μg/ml (F) in
the presence of LPS, for 24 hrs. The cells were then fixed and stained in oil red O solution. (×400).
(Fig. 1B and Fig. 5).
In contrast, Macrophages secreted anti-inflammatory cyto-
kines after treatment with cordycepin or adenosine (Fig. 3),
which differentiated M2 macrophages (Fig. 1). M2 macropha-
ges are generally characterized by low production of pro-in-
flammatory cytokines and high expression of anti-inflamma-
tory mediators such as IL-1ra, IL-10, and TGF-
β
(Fig. 3). Fur-
ther research will be needed to examine IL-1ra expression
which was down-regulated by cordycepin (Fig. 3A) versus
adenosine, which showed an up-regulation of anti-inflamma-
tory cytokines (Fig. 3B). Cells treated with both cordycepin
and adenosine still induced the M2 phenotype from the phe-
notype of macrophages when treated with LPS only (Fig. 4).
The purpose of this study was to investigate the effects of
the main constituents of
Cordyceps militaris
, cordycepin and
adenosine, on macrophage maturation/differentiation and cy-
tokine production as well as to determine how the immune
responses are modulated in inflammatory diseases. We dem-
onstrated that both cordycepin and adenosine were able to
regulate immune responses by producing anti-inflammatory
cytokines and also showed the phenotypic switch from M1
to M2 in macrophages. Treatment with both cordycepin and
adenosine in LPS-activated macrophages was examined and
resulted in a down-regulation of M1 cytokines and chemo-
kines and alternatively, an up-regulation of M2 cytokines in
macrophages.
ACKNOWLEDGEMENTS
This paper was supported by the Sahmyook University
Research fund, 2009.
CONFLICTS OF INTEREST
The authors have no financial conflict of interest.
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