Extract
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Introduction
Artemisinin is extracted from the herb Artemisia annua
L, and various forms of the drug are used as anti-malarial
agents[1,2]. Artemisinin is a potent anti-malarial agent with
low toxicity, and its derivatives have improved efficacy in
malaria treatment relative to artemisinin itself. In addition to
anti-malarial effects, anti-lymphocytic effects have also been
reported for artemisinin
derivatives[3,4]. Since the 1980s, the
immunosuppressive actions of artemisinin and its
derivatives have been studied in China. Artemisinin derivatives
have also been tested for the treatment of dermatoses such
as photoallergic skin diseases and systemic lupus
erythema-tosus, and promising results have been
reported[5-7].
Researchers have demonstrated that the anti-malarial
activity of artemisinin and its derivatives is associated with
their endoperoxides[8,9]. When catalyzed by the ferrous ion
in heme, cleavage of peroxy bonds produces free radicals
and thus causes damage to malarial DNA and proteins.
However, the underlying mechanism of the anti-lymphocytic
action of artemisinin and its derivatives is poorly understood.
Despite their anti-lymphocytic mechanisms being poorly
understood, we hypothesized that artemisinin derivatives
might be promising immunosuppressive agents, because
some clinical studies have shown that they are effective in
the treatment of immune dysfunction diseases. When
considering an optimal molecular structure (higher efficacy and
lower toxicity) for artemisinin derivatives for use as
immuno-suppressants, we noticed that nonsteroidal
anti-inflammatory drugs (NSAIDs) remained the first-line therapy for most
people with arthritis and other inflammatory diseases.
Compared with other anti-inflammatory or immunosuppressive
drugs such as steroids and cyclosporine, the molecular
structures of NSAIDs are relatively simple and easy to synthesize.
In addition, NSAIDs have few side effects, and a low level of
toxicity, which are clinically desirable features. Given the
relevance of the structure of NSAIDs to their
immunosuppressive activity, we became interested in combining
artemisinin and NSAIDs to develop a new class of
immunosuppressive agents. Therefore, we synthesized and studied
more than 100 different artemisinin derivatives. As a result
of this screening program and subsequent medicinal
chemistry studies, we obtained some derivatives that have
significant immunosuppressive activity. Previous studies
indicated that SM735 ([3-(12-b-artemisininoxy)]phenoxyl succinic
acid,
C25H32O9·1/2H
2O, MW: 485.5, Figure 1) is a representative compound, with markedly lower toxicity and higher
immunosuppressive activity than
artemisinin[10]. Here, we further investigate its immunological characteristics both
in vitro
and in vivo.
The discovery of cyclosporine (cyclosporin A; CsA) and
its successful utilization in organ transplantation was a
milestone in clinical transplantation. CsA has produced a new
era in transplantation in terms of both efficiency and quality
of life for patients. In addition, research into the
mechanisms by which CsA acts has been rewarding in that we now
have a better understanding of the mechanisms leading to T
lymphocyte activation[11,12]. Recently, Noori
et al reported that the immunosuppressive activity of artemisinin was even
greater than that of CsA, as indicated by both in
vivo and in vitro
studies[13]. Therefore, in the present study, we
demonstrate that SM735 has strong immunosuppressive effects
in vitro and in vivo, and explore its possible mechanism of action.
Materials and methods
Reagents Concanavalin A, lipopolysaccharide
(Escherichia coli 055:B5), ionomycin, phorbol 12-myristate
13-acetate (PMA),
3-[4,5-dimethylthylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), and 3,3¡¯,5,5¡¯-tetramethyl-
benzidine were purchased from Sigma (St Louis, MO, USA).
Mitomycin-C was purchased from Kyowa Hakko (Tokyo,
Japan). RPMI-1640 and fetal bovine serum (FBS) was
purchased from Gibco (Life Technology, NY, USA).
1-Fluoro-2,4-dinitrobenzene (DNFB) was purchased from Merck (Hong
Kong, China). Mouse enzyme-linked immunosorbent assay
(ELISA) kits of interleukin (IL)-2, IL-12p40, interferon
(IFN)-g and IL-6 were purchased from Pharmingen (San Diego, CA,
USA).
Cyclosporine (Sandimmun) was purchased from Novartis
Pharma AG, Switzerland. Cyclophosphamide was purchased
from Hualian Pharma (Shanghai, China). SM735
[3-(12-b-artemisininoxy)phenoxyl succinic acid] was synthesized by
us. Before use, SM735 was dissolved in pure dimethyl
sulfoxide (Me2SO; 100 g/L) as a stock solution, and stored at
4 oC. The stock solution was diluted to the needed
concentrations with RPMI-1640 supplemented with 10% FBS. The
final concentration of Me2SO in the culture medium was less
than 0.01%, which had no influence on the
assays[10,14]. For in vivo experiments, both SM735 and the reference drugs
were dissolved in 0.5% Tween-80, 0.33%
Me2SO in saline.
Animals and housing conditions Inbred 7-9-week-old
BALB/c, C57BL/6 mice were obtained from the Shanghai
Experimental Animal Center of the Chinese Academy of
Sciences (Certificate No 99-003). The mice were housed in
specific pathogen-free conditions with a room temperature of
24±2 oC, a 12-h light/dark cycle, and provided with sterile
food and water ad libitum. All mice were allowed to acclima
tize in our facility for 1 week before any experiments were
started. All experiments were carried out according to the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals, and were approved by the Bioethics
Committee of the Shanghai Institute of Materia Medica.
Preparation of splenocytes Splenocytes were prepared
aseptically from inbred 7-9-week-old BALB/c, C57BL/6 mice,
and cultured in RPMI-1640 supplemented with 10%
endotoxin-free, heat-inactivated FBS, 100 kU/L penicillin, 100 mg/L
streptomycin, 10 mmol/L
N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid (HEPES), and 50 µmol/L 2-mercapto-
ethanol (2-ME).
Lymphocyte proliferation and cytotoxicity
assay
Lymphocyte proliferation was carried out as described
elsewhere[15]. Briefly, 7-8-week-old male BALB/c mice were killed
and splenocytes were prepared aseptically to a single-cell
suspension. Splenic lymphocytes were stimulated with ConA
(5 mg/L) or lipopolysaccharide (LPS 10 mg/L), plus the
required concentrations of drugs. The cell cultures were then
incubated for 48 h at 37 oC in a humidified 5%
CO2 incubator. Cells were pulsed with 0.5 µCi/well
[3H]-thymidine 8 h prior to the end of the culture. After 48 h, cells were harvested
onto a glass fiber filter using a
HARVESTER96Ò (TOMTEC ,USA) 96-well cell harvester and incorporated radioactivity
was counted with a Beta Scintillation counter (1450
MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA,
USA ).
Cytotoxicity was assessed by using the MTT assay.
Briefly, 15 µL of 5 g/L MTT was pulsed 4 h prior to the end of
the culture (in a total volume of 160 µL), and then 80 µL
solvent [10% sodium dodecylsulfate (SDS), 50%
N,N-dimethyl formamide, pH 7.2] was added to dissolve the precipitate.
The solution was incubated for another 7 h and
OD570 nm was read by using a microplate reader (Bio-Rad, Model 550,
Tokyo, Japan).
Mixed lymphocyte reaction proliferation assay The
mixed lymphocyte reaction (MLR) was carried out as
previously described, with some
modifications[16]. Briefly, spleen cells from 7-9-week-old BALB/c mice were prepared in a
1×1010 cells/L suspension, which was inactivated for 2 h with
50 mg/L of mitomycin C. Cells were washed and co-cultured
with freshly prepared splenocytes from C57/BL6 mice (the
ratio of stimulator to responder was 1.0) in a final
concentration of 1.5×109 cells/L for 96 h. The desired concentrations
of each compound were added in cultures for immunological
activity assays. [3H]Thymidine (0.5 µCi/well) was pulsed 24
h prior to the end of the culture and then cells were harvested
onto a glass fiber filter for measurement of incorporated
radioactivity.
Cytokine production and analysis Splenocytes
(5×106) were prepared from 7-9-week-old BALB/c mice, added to
1 mL of RPMI-1640 media, and then were incubated with 5
mg/L of ConA, 10 mg/L of LPS, or 10 µg/L of PMA plus 1
µmol/L of ionomycin, for 24 h of culture in 48-well
microculture plates. The supernatants were harvested and stored
at -80 oC before the assay was carried out. Cytokine
levels in supernatants were measured by ELISA, according to
the manufacturer¡¯s instructions (Pharmingen, San Diego, CA,
USA). 3,3¡¯,5,5¡¯-Tetramethylbenzidine was used to develop
the color reaction. The absorbance was read at 450 nm by a
microplate reader (Bio-Rad, model 550, Tokyo, Japan).
Cytokine concentrations were calculated based on a
standard curve created using standard murine cytokines.
DNFB-induced delayed-type hypersensitivity
response Female BALB/c mice were randomized into 6 groups
and sensitized with 20 µL of 0.5% DNFB dissolved in
acetone-olive oil (4:1) on each hind foot on d 0 and d 1.
Me2SO vehicle, CsA and SM735 were administered to each group
(n=10) by intraperitoneal injection on 4 consecutive days
(d 7-d 10). On d 9 mice were challenged with 10 µL of 0.2%
DNFB on both sides of the right ear, using the method
described by Phanuphak et al, with some
modifications[17]. The extent of ear swelling was expressed as the difference
between the weight of punches taken from the left and right
ears by using an 8-mm punch 48 h after the second challenge.
Quantitative hemolysis of sheep red blood
cells Female BALB/c mice were immunized by ip injection with 0.2
mL of 16.7% sheep red blood cells (SRBC) on d 0.
Me2SO vehicle, 25 mg/kg of cyclophosphamide and SM735 were
administered to each group (n=6) by ip injection on 4
consecutive days (d 1-d 4). On d 5, mice were killed, and mixed
suspensions of 2×109 spleen cells/L were made. A total of 1
mL of cell suspension was incubated with 1 mL of 0.5% SRBC
and 1 mL of 1:10 dilution of guinea pig complement for 0.5 h
at 37 oC. The suspension was then centrifuged (3 min at
3000×g) and the extent of hemolysis in the supernatant was
determined at 520 nm, according to the method used by
Simpson and Gozzo with some
modifications[18].
Statistical analysis Three independent experiments were
performed with similar results. We counted the 50%
cytotoxic concentration (CC50) and the 50% inhibitory
concentration (IC50) values using the Origin software package (Microcal
Software). Student¡¯s t-test and one-way analysis of
variance (ANOVA) with Newman-Keuls multiple comparisons
on post-tests were used to analyze data and compare groups.
P<0.05 was considered significant.
Results
Cytotoxicity of SM735 for murine splenocytes To
determine the safe dose range of SM735, we first examined
the cytotoxic effect of the compound. As Figure 2A shows,
in a 48-h culture, SM735 had a typical S-shaped
concentration-toxicity relationship for murine splenocytes. SM735 at
a dose of 25 µmol/L showed no cytotoxicity (inhibitive rate
<10%, P>0.05). The 50% cytotoxic concentration
(CC50) value of SM735 was 53.1±7.8 µmol/L for a 48-h culture.
SM735 inhibits mitogen-induced murine splenocyte
proliferation Concentration-dependent suppression of
murine splenocyte proliferation in response to ConA and LPS
was observed when SM735 was added to cell cultures.
Figure 2B, 2C shows that proliferation induced by ConA or LPS
was significantly inhibited by SM735 exposure. Both
concentration-dependencies fitted typical S-shaped curves.
Comparisons with a vehicle control showed that this effect
was statistically significant (P<0.05) when concentrations
of SM735 were above 0.04 µmol/L in response to ConA and
above 0.02 µmol/L in response to LPS. In response to ConA,
the IC50 value of SM735 inhibition of lymphocyte
proliferation was 0.33±0.06 µmol/L, and in response to LPS, the
IC50 value was 0.27±0.02 µmol/L.
SM735 inhibits splenocyte proliferation in MLR
Mitomycin C-inactivated splenocytes from BALB/c mice
(H-2d) were applied as allogeneic stimuli to proliferating splenocytes
of C57BL/6 mice (H-2b). As shown in Figure 2D, SM735
strongly suppressed T cell proliferation in MLR in a
dose-dependent manner, with an IC50 value of 0.86±0.18 µmol/L
for a 96-h co-culture. When concentrations of SM735 were
above 0.08 µmol/L, comparisons with the vehicle control
showed that this effect was statistically significant
(P<0.05). The IC50 values were comparable to those when
ConA-stimulated T cell proliferation was suppressed.
Effects of SM735 on cytokine production We used ConA
alone, LPS alone, or PMA plus ionomycin as stimuli to
promote cytokine secretion in mouse splenocytes. Then the
effects of SM735 on the production of proinflammatory
cytokine IL-6, and Th1-type cytokines IL-2, IL-12, and
IFN-g, were examined. The results are summarized in Table 1. The
production of IL-12, IFN-g, and IL-6 was significantly
decreased in a concentration-dependent manner when the
splenocyte cultures were exposed to SM735, upon
stimulation with LPS or PMA plus ionomycin. We also detected a
strong inhibitive effect of SM735 on IFN-g production
induced by ConA. Unexpectedly but interestingly, we did not
observe a significant effect of SM735 on IL-2 production.
SM735 suppresses DNFB-induced delayed-type hy
persensitivity reaction Figure 3 illustrates the
dose-dependent inhibitory effect of SM735 on DNFB-induced DTH ear
swelling. When SM735 was administered for 4 consecutive
days, at doses of 7.5, 15, and 30 mg/kg, SM735 significantly
suppressed ear swelling by 19.6%, 35.5%, and 55.4%,
respec-tively. The inhibition was comparable to that of CsA, which
exerted 55.2% suppression at a dose of 50 mg/kg.
SM735 suppresses anti-SRBC specific immunoglobulin
production Quantitative hemolysis of SRBC (QHS) is
a model of primary antibody production in response to
antigenic stimulation. Because CsA specifically blocks T cell
activation by suppressing IL-2 production, with little effect
on B cells, we chose cyclophosphamide (CTX) as a
reference drug in the present study[19]. As Figure 4 shows,
administration of 15 and 30 mg/kg SM735 for 4 consecutive
days significantly suppressed QHS in a dose-dependent
manner. The inhibitive rates were 15.3% (P<0.05) and 25.4%
(P<0.01), respectively, slightly less potent than the 31.8%
inhibition effected by CTX (P<0.01).
Discussion
Patients who receive organ grafts are principally treated
with immunosuppressive agents. In addition, there are a
number of other autoimmune chronic inflammatory disorders
that might be able to be treated with immunosuppressive
drugs. In the 1970s and 1980s, the discovery and clinical use
of CsA and tacrolimus (FK506) enabled the successful
transplantation of major organs in human patients. However, it
seems now that neither are able to be tolerated in the long
term[20,21]. These drugs cause systemic immunosuppression
that greatly increases the risks of tumors arising and lethal
fungal infections occurring. Therefore, new
immunosuppressants are required that possess better therapeutic effects or
can be combined with currently used drugs to reduce
long-term tolerance and side effects.
Previous studies have indicated that artemisinin
possesses anti-lymphocytic activities. There are numerous
reports of clinical cases in which autoimmune diseases have
been treated with artemisinin and artesunate; therapeutic
effects were achieved in these cases, thus encouraging us
to study artemisinin and its derivatives. To improve the
efficacy of the drug, we optimized artemisinin by linking it
with various NSAID structures. Here, we report that a newly
synthesized artemisinin derivative, SM735, possesses
potent immunosuppressive activity both in
vitro and in vivo.
Lymphocyte proliferation is a crucial event in the
activation cascade of both cellular and humoral immune responses.
Treatment with SM735 can dose-dependently reduce both
the T and B cell proliferation promoted by mitogens.
Because it has a relatively high CC50 value, SM735 has a
favorably large safety range.
A mixed lymphocyte reaction is induced by allogeneic
stimuli, and T lymphocytes are the main responders,
indicating an immune response very similar to that which occurs in
post-transplantation graft-versus-host
diseases[22,23]. MLR is often used clinically for tissue typing to identify the
compatibility of donor organs and recipients. Furthermore, the
suppression of MLR by immunosuppressants also helps
improve the success of
transplantation[24,25]. Here we show that SM735 also potently inhibited MLR, with an
IC50 value comparable to that for the inhibition of T cell proliferation.
At concentrations above 10 µmol/L, SM735 almost
completely abrogated MLR. This suggests that SM735 would
probably act as an immunosuppressant in physiological
conditions.
Cytokines are important modulators and effectors in the
immune system. In particular, multiple proinflammatory
cytokines have been proved to be closely associated with
many autoimmune diseases. It is critical to silence
proinflam-matory cytokines to maintain successful
immunosuppression[26-28]. In the present study, we used mitogen ConA,
bacterial LPS, and PMA plus ionomycin, which agitated the
whole cell population by activating protein kinase C, to
promote cytokine production in splenocytes. The
results showed that SM735 significantly inhibited IL-12,
IFN-g and IL-6 production with LPS or PMA plus ionomycin stimulation. When
using ConA as a stimulant, we also observed a
dose-dependent inhibition of IFN-g production. Unexpectedly, the IL-2
level was not obviously altered. Although the two
cornerstone immunosuppressants, CsA and FK506, are putatively
regarded as IL-2 inhibitors, it seems that SM735 acts totally
differently, on some event(s) downstream of IL-2-mediated
naive T cell activation. IL-2 is widely considered to be a key
cytokine in T-cell-dependent immune responses. However,
the main non-redundant activity of this cytokine centers on
the regulation of T cell tolerance, and recent studies have
indicated that a failure in the production of
CD4+CD25+ regulatory T cells is the underlying cause of autoimmunity in the
absence of IL-2[29]. Thus, the fact that SM735 does not
inhibit IL-2 indicates that further research is necessary on the
induction of long-term immune tolerance by SM735.
To examine the immunoregulatory effects of SM735
in vivo, we used a mouse DTH model and the QHS model. Ear
swelling in DTH is primarily the result of antigen-specific
CD4+ T cell activation[30]. Administration of SM735 for 4
consecutive days significantly suppressed ear swelling,
indicating that SM735 is capable of inhibiting the T-cell-de
pendent immune response in vivo. In our experiments, the
effect of SM735 was comparable to that of cyclosporine at a
curative dose. The QHS model reflects the
antibody-producing capacity of plasma cells in response to
SRBC[18,19]. The suppressive effect of SM735 in QHS indicated that it
also suppressed antibody-secreting B cells in
vivo.
In conclusion, our results demonstrate that SM735, a
newly synthesized artemisinin derivative, has strong
immunosuppressive effects in vitro and in
vivo. Because artemi-sinin and its derivatives potentially have low toxicity and
few side effects[4,31], our study suggests the possibility of
developing SM735 and other artemisinin derivatives as novel
safe immunosuppressants, probably with a different
mechanism from that of CsA or FK506. However, further studies
are necessary to elucidate the details of the mechanism.
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