Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 445-452
doi:10.1111/j.1745-7270.2007.00300.x
A Lectin from Chinese Mistletoe Increases gd T Cell-mediated Cytotoxicity through
Induction of Caspase-dependent Apoptosis
Fang GONG1, Yanhui MA1, Anlun
MA1,3, Qiwen YU1, Jiying ZHANG1, Hong
NIE1, Xuehua CHEN2, Baihua SHEN1, Ningli LI1, and Dongqing ZHANG1*
1 Shanghai Jiaotong
University School of Medicine, Shanghai Institute of Immunology, Shanghai
200025, China;
2
Shanghai
Institute of Digestive Surgery, Ruijin Hospital, Shanghai 200025,China;
3 Centre de Recherche du CHUM,
H?pital Notre-Dame, Universit? de Montr?al, Montr?al, Qu?bec H2L 4M1, Canada
Received: February
12, 2007
Accepted: April 18,
2007
This work was
supported by the grants from the National Natural Science Foundation of China
(No.30471593, No.30670939), the Shanghai Leading Academic Discipline Project
(T0206), and the Shanghai Institute of Immunology Project (07-A02)
*Corresponding
author: Tel, 86-21-64453049; Fax, 86-21-64453049; E-mail,
Abstract In this study, a mistletoe lectin (ML) was purified from
Chinese mistletoe and the effect of this 60 kDa Chinese ML on human gd T cell
cytotoxicity, apoptosis and modulation of the cytokine network was studied. The
cytotoxic properties of d T cells was evaluated by using a 51Cr
release test and employed fluorescence-activated cell sorting and enzyme-linked
immunosorbent assay analysis to quantify translocation of the cell membrane
phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis.
It was found that: (i) ML effectively stimulated gd T cell proliferation in a
dose- and time-dependent manner; (ii) ML increased gd T cell cytotoxicity;
(iii) ML could modulate lipopolysaccharide-induced cytokine release in a
pro-inflammatory manner by increasing tumor necrosis factor (TNF)-a release and
inhibiting the release of anti-inflammatory interleukin (IL)-10; (iv) ML
induced apoptosis in caspase-dependent and CD95-independent manner. The
results indicated that ML is a potent immunomodulator to human gd T cell
cytotoxicity, apoptosis and cytokine production.
Keywords mistletoe lectin; gd T cell; cytotoxicity; apoptosis; cytokine
Mistletoe (Viscum album), a semiparasitic plant, is an unusual
plant with many unusual properties. It was used as a herbal remedy in the
ancient Chinese Pharmacopoeia and has been used in traditional Chinese medicine
for diseases such as gonorrhea, syphilis, hypertension and rheumatism for
thousands of years. The aqueous extract of European mistletoe has been used in
conventional cancer therapy for decades [1]. Therapeutic efficacy is mostly
attributed to the mistletoe lectins (MLs), ML-I, ML-II and ML-III, which belong
to the “toxic lectin family” and represent ribosome-deactivating
proteins class II. They consist of an N-glycosidase (A chain) and a
galactosid-binding lectin (B chain) linked by a disulfide bridge. The lectins
ML-I and ML-III preferably bind to galactosid- or N-acetylgalactosamin-groups
while ML-II can bind to both carbohydrates [2].MLs have recently been found to act through several distinct
bioactivities as potent immunomodulators. First, MLs exerted a broad
immunostimulatory activity by activating different types of cells [35] in cell
cultures and animal models. Incubation of lymphocytes with ML-I could result in
antitumoral cytotoxic T lymphocytes bearing phosphorylated mistletoe ligands
[6,7]. Second, MLs favored bridging of natural killer-tumor cell conjugates,
enhancing efficiency of killing [8–10]. Third, it has been found that MLs could
activate immune responses by modulating the complex network of cytokines that
regulate leukocyte functions. ML-I caused an increased secretion of tumor
necrosis factor (TNF)-a, interleukin (IL)-1, and IL-6 from isolated human mononuclear cells
in vitro [11,12]. Finally, MLs have been described as inducers of
apoptosis. In the presence of ML-I, human mononuclear cells and many cell lines
[1,13] underwent apoptosis.While the European mistletoe has been studied intensively, less is
known about the Chinese mistletoe as a anticancer drug. In our present study, a
protocol for Chinese mistletoe extract preparation was introduced. The aims of
this study were to evaluate the potency of the ML from Chinese mistletoe on human gd T cell cytotoxicity, apoptosis induction and modulation of the
cytokine network in vitro.
Materials and Methods
Reagents
Lipopolysaccharide (LPS), concanavalin A (Con A), phytohemagglutinin
(PHA), methanol, ethanol, cyclohexane, dichloromethane, ethyl acetate,
CM-Sepharose, sodium dodecyl sulfate (SDS)-polyacrylamide gel and Coomassie
blue dye were obtained from Sigma (St. Louis, USA). Na251CrO4 was obtained from ICN Biochemicals (Costa Mesa, USA). CD95, gd T cell
receptor (TCR), IL-10, Annexin V fluorescein-isothiocyanate (FITC) monoclonal
antibodies, caspase inhibitor zVAD-fmk, and propidium iodide (PI) were
purchased from Becton Dickinson (Mountain View, USA). The gd TCR magnetic
cell isolation kit was purchased from Miltenyi Biotec (Bergisch Gladbach,
Germany). The cell death detection kit was obtained from Roche (Indianapolis,
USA). The TNF and IL-10 enzyme-linked immunosorbent assay (ELISA) kits were
obtained from R&D Systems (Minneapolis, USA).
Preparation of mistletoe extract
ML was isolated from water extracts of Chinese mistletoe, a
subspecies of V. album according to the previous methods [14] with our
own modifications. Briefly, the air-dried mistletoe (3 kg), collected from
Sichuan province, China, was crushed and extracted twice with 20 liters of
methanol/water (1:1, V/V). The homogenate was filtered through a
nylon cloth. After filtration, with its volume reduced to 2 liters, the aqueous
phase was successively partitioned with cyclohexane, dichloromethane and ethyl
acetate. Ethanol was added to the concentrated aqueous phase to a final
concentration of 85% (V/V). A precipitate was obtained and
separated from the supernatant by centrifugation (8000 g, 20 min). The
supernatant was concentrated and ethanol was added to 85% (V/V).
After centrifugation, the precipitate was collected and combined with the
former precipitate. The final yield of ML extract was 100 g from 3 kg
mistletoe. All the precipitate was dissolved in 100 ml of phosphate buffer (10
mM, pH 6.5) and the stock solution of mistletoe extract was stored at –80 ?C.
Purification of ML
Purification of ML
To obtain the pure ML, mistletoe protein extract was further
purified by CM-Sepharose column chromatography [14]. The aqueous layer (1 ml) was
applied to a column of CM-Sepharose (1.5 cm20 cm) equilibrated with 10 mM
phosphate buffer (pH 6.5). After washing with 10 mM phosphate buffer (pH 6.5)
and 100 mM NaCl in the same buffer at a rate of 0.5 ml/min, a peak eluted with
500 mM NaCl in the same buffer was dialyzed with phosphate buffered saline
(PBS) (pH 7.4). The fractions containing hemagglutinating protein were
collected and then applied to a column of Con A column (1.5 cm20 cm)
equilibrated with 10 mM PBS (pH 7.4). The column was washed with PBS (pH 7.4)
and eluted with 300 mM glucose in the same buffer. Fractions were subject to
SDS-electrophoresis and fractions containing 60 kDa protein were pooled,
dialyzed against water and freeze-dried.
SDS-PAGE
The molecular mass and purity of ML was determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 12% polyacrylamide gel
was used as resolving gel and 5% was used as stacking gel. To further denature
the proteins by reducing disulfide linkages, the samples were heated at 100 ?C
for 3 min in the presence of a reducing agent. The samples were
electrophoresed using electrophoresis system at 200 V for 75 min and gels were
stained with Coomassie brilliant R-250.
Cell lines
Human T lymphoma Jurkat, Burkitt’s lymphoma Daudi (CD95 sensitive)
and Jurkat-R (CD95 resistant) cell lines were purchased from American Type
Culture Collection (Manassas, USA). The cells were cultured and maintained in
complete RPMI 1640 at 37 ?C in 5% CO2 in an incubator. The
CD95-resistant Jurkat subline Jurkat-R was generated by continuous culture in
the presence of anti-CD95 antibody.
Cytotoxin T lymphocyte assay
Purified human gd T cells with magnetic activatited cell sorting were used as
effectors. Daudi tumor cells used as targets were labeled with 100 mCi of Na251CrO4, washed and plated out at a concentration of
5103 cells/well in 96-well round bottom culture plates. The effector
cells were added to achieve effector : target (E:T) ratios of 1000:1, 100:1 and
10:1. The percentage of specific 51Cr release was measured after
the plates had been incubated for 4 h at 37 ?C in 5% CO2 in air.
Maximum 51Cr release was measured by osmotic lysis of the cells. The gd T
cell-mediated cytotoxity was calculated according to the following formula:
Eq.
Quantification of cytokine production
The production of IL-10 and TNF cytokine was quantified by ELISA
assay according to the manufacturer’s protocol. Human peripheral blood mononuclear
cells (PBMCs) were plated in 24-well plates at 106
cells/well in the presence of ML, and the supernatant was harvested after 24 h
and 72 h of incubation. A590 were measured by using a
plate reader (Molecular Devices, Wilmington, USA).
Flow cytometry and detection of apoptosis
Cells were washed twice with cold PBS then resuspended in 1?binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1106/ml. The solution (100 ml) was
transferred to a 5 ml culture tube, with 5 ml of Annexin V-FITC and 5 ml of PI added.
Cells were gently vortexed and incubated for 15 min at room temperature in the
dark. Then 400 ml of binding buffer was added and cells were analyzed by flow cytometry
within 1 h. Cells that stained positive for Annexin V-FITC and negative for PI
were undergoing apoptosis. Cells that stained positive for both Annexin V-FITC
and PI were either in the end stage of apoptosis, were undergoing necrosis, or
were dead. Cells that stained negative for both Annexin V-FITC and PI were
viable and not undergoing measurable apoptosis.To analyze the time dependency of apoptotic DNA fragmentation, cells
were plated in 96-well plates, and 24 h later, ML (100 ng/ml and 1000 ng/ml)
was added to the culture medium and incubated for an additional 24, 48 or 72 h.
Apoptosis was measured in triplicates using the cell death detection kit.
Results
SDS-PAGE
Chinese ML extractions were analyzed by SDS-PAGE. In the presence of
the reducing agent, it showed an estimated 60 kDa band consisting of two
bands of a 30 kDa A chain and a 34 kDa B chain (Fig. 1).
ML induces proliferation of gd T cells
To evaluate the stimulatory activity of ML on human gd T lymphocyte
proliferation, PBMCs treated with different concentrations of ML (0.1–2000 ng/ml) were
cultured for 72 h. The data shows that ML at low concentrations
did not cause a significant increase of gd T cells [Fig.
2(A)], however, levels up to 45% of gd T cells were
observed in the PBMCs activated with ML 2000 ng/ml, compared to 4.2% of the
control group. When the ML concentration ranged from 100 ng/ml to 2000 ng/ml,
the population of gd T cells proliferated in a dose-dependent manner. A similar
phenomenon was also observed in PHA-treated cells [Fig. 2(A)]. The time
curves were obtained in ML- and PHA-stimulated gd T cell
proliferation at 12, 24, 48 and 72 h [Fig. 2(B)]. We conclude that ML
can stimulate the proliferation of gd T cells in both a dose- and
time-dependent manner.
ML enhances cytotoxicity of gd T cells
To verify whether ML exerted cytotoxicity by activating gd T cells,
cells from the continuously-growing malignant human Burkitt’s lymphoma Daudi
cell (natural killer-resistant) line were chosen as the target. gd T cells
pre-treated with ML (1000 ng/ml) as effector cells were added to achieve E:T
ratios of 1000:1, 100:1 and 10:1. This study found that, without gd T cells,
the spontaneous release of 51Cr from
target cells alone with ML was not directly toxic to these targets (<5%, data not shown). At an E:T ratio of 1000:1, in the presence of 1000 ng/ml ML, the cytotoxicity of gd T cells was 6.83, 9.43 and 3.84 times higher
than in the presence of 1 ng/ml ML at 24, 48 and 72 h, respectively [Fig.
3(A)]. Similar results were also found at E:T ratios of 100:1
and 10:1 [Fig. 3(B,C)]. The level of cytotoxicity was shown to be
dependent on the number of effector cells and the ML concentration used.
ML shifts cytokine secretion in PBMCs
Stimulation of PBMCs with LPS-free ML did not cause any significant
cytokine release (data not shown). Similar results were also found at low concentrations ranging from 1 to 10 ng/ml ML. However, when treated
with 1001000 ng/ml ML, the LPS-induced cytokine secretion shifted
toward a pro-inflammatory response. The release of TNF-a was higher than
the LPS control [Fig. 4(A)]. In the same concentration range, ML caused a diminished secretion of IL-10
compared with the LPS control [Fig. 4(B)].In the presence of ML (1000 ng/ml), the time curve for TNF-a release in
LPS-stimulated PBMCs peaked at 8 h of incubation then showly decreased. At 24
h, the level was approximately 30.4% lower. Similar kinetics of TNF formation
were also observed when a neutralizing antibody against IL-10 was added to
LPS-stimulated PBMCs. Here, the initial formation of TNF-a was not
affected, and anti-IL-10 decreased the levels of TNF-a after 8 h of
incubation [Fig. 4(C)].
ML induces caspase-dependent apoptosis
One of the earliest indications of apoptosis is the translocation of
the membrane phospholipid phosphatidylserine (PS), which becomes available for
Annexin V with a high affinity. However, PS translocation also occurs during
necrosis, so PI is often used to bind to nucleic acids. To identify the mode of
the apoptosis, we treated Jurkat leukemic T cells
with ML (100 ng/ml and 1000 ng/ml) for 24 h. Fig. 5 shows the effect of
Annexin V-FITC/PI that discriminated between apoptotic and necrotic cells. The
apoptotic cell count after 24 h treatment with ML was comparable with that
observed in the untreated control cells. Annexin V positive cells were found at
levels of (35.277.6)% and (54.1410.9)% in Jurkat cells treated with 100 ng/ml
and 1000 ng/ml ML, respectively [Fig. 5(B,C)], compared with control
cells of (5.001.89)% [Fig. 5(A)], and increased in a dose-dependent
manner. To confirm apoptosis, we used an ELISA-based cell death detection
assay. The maximum effect occurred 48 h after the treatment. In addition, if ML
induced a specific apoptosis, then DNA fragmentation should be partially or
fully prevented in cells co-treated with ML and caspase family inhibitors. To
address this question, we co-treated Jurkat cells with ML (100 ng/ml) and
zVAD-fmk (100 ng/ml), a general upstream inhibitor of caspases. The inhibitor
was replenished daily when the treatment exceeded 24 h. Our results [Fig.
5(D)] showed that zVAD-fmk fully decreased ML-induced DNA fragmentation and
the caspase family inhibitor could protect ML treated cells from apoptotic
death.
ML induces CD95-independent apoptosis
To investigate whether the CD95 receptor/ligand interaction was
involved in ML-induced apoptosis, we used the subclone Jurkat-R cells, which
were resistant to the CD95 signal. When CD95-sensitive Jurkat and
CD95-resistant Jurkat-R cells were treated with 1000 ng/ml ML, both cell lines
underwent apoptosis with a very similar dose-dependency (data not shown). In
contrast, an agonistic anti-CD95 antibody induced apoptosis in Jurkat cells, but
not in Jurkat-R cells, confirming that these cells were, indeed, CD95-resistant
[Fig. 5(E)].
Discussion
Extracts from European mistletoe are widely used in the treatment of
cancers, but the mechanism of antitumor properties has not yet been clearly elucidated
[1]. In this respect, we prepared and purified a Chinese ML and analyzed its
antineoplastic activity. In SDS-PAGE, it showed a estimated 60 kDa band
consisting of two bands of a 30 kDa A chain and a 34 kDa B chain, whereas
European mistletoe lectin shows two bands of 32 kDa and 27 kDa [15]. Our
results showed a 60 kDa Chinese ML displayed as an activator on the
proliferation of gd T cells in both a time- and concentration-dependent manner.
It also appeared that it could enhance the cytotoxicity of gd T cells.
Furthmore, our results showed that purified LPS-free mistletoe preparations did
not induce cytokines in human PBMCs. Therefore, we conclude that 60 kDa Chinese
ML has no pyrogenic activity [13,16]. Surprisingly, it was found to modulate
LPS-induced cytokine release in a pro-inflammatory manner by increasing TNF-
release and inhibiting the release of anti-inflammatory IL-10. The reduction
of IL-10 production is likely to affect further immune responses besides TNF-a formation in a
pro-inflammatory manner. After LPS stimulation of PBMCs, lymphocytes release
considerable amounts of TNF- within a few hours. TNF-a release is then
down-regulated by the secretion of its endogenous antagonist IL-10 [17]. In
this feedback loop, IL-10 inhibits the inflammatory cytokines like TNF-a, probably
through the inhibition of the nuclear factor kB [18]. The mechanism by which Chinese MLs exert their IL-10-inhibiting effects remains to be clarified. Our results provide the evidence that 60 kDa Chinese
ML interferes with and modulates the cytokine network of
LPS-stimulated PBMCs in a pro-inflammatory manner,
and will encourage further study to clarify possible
beneficial effects.As different approaches have shown the activation of the apoptotic
program, we tried to elucidate the mechanism of this activation involved in
the anti-tumor effects of the Chinese ML. Initiation of the apoptosis response
involves “initiation” caspases, such as caspase 8, which might induce
apoptosis directly by activating “effector” caspases such as caspases
3, 6 and 7 [19,20]. To investigate the nature of the selective cell death
induced specifically by the action of Chinese ML, Jurkat cells were analyzed
for apoptosis by exposure to PS and DNA fragmentation, which are typical signs
of apoptosis activation. In the present study, a quantitative estimation of the
percentage of Chinese ML inducing apoptosis gave values of approximately 35%
of apoptotic cells and these results were further confirmed by ELISA. These
results also showed that induction of apoptosis by Chinese ML was entirely
dependent on the intracellular activation of caspases, because cell death was
completely prevented by zVAD-fmk, a broad caspase inhibitor. The study suggests
that 60 kDa Chinese ML-triggered cell death is caspase-dependent and plays an
important role in its anti-tumor activities.In the death receptor pathway, caspase 8 is a crucial component of
the death-inducing signaling complex, where it is recruited through its
Database of Evolutionary Distances (DEDs) that interacts with Fas-associated
protein with death domain [2123]. The activation of caspase 8/FLICE seems to
be restricted to apoptosis mediated by death receptors including CD95, TNF
receptor 1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors.
Because caspase 8 is one of only two databases of evolutionary
distances-containing proteases, it is assumed to act as a proximal initiator
caspase, which subsequently processes downstream effector caspases. Initially,
the activation of caspase 8, therefore, led us to suggest that death receptors
such as CD95 might be involved in ML-induced apoptosis. It could be speculated
that Chinese ML induces the expression of CD95 ligand, resulting in subsequent
CD95-dependent apoptosis by an autocrine or paracrine mechanism. Such a
scenario has been previously proposed for apoptosis mediated by anticancer
drugs in some experimental systems [2426]. Another possibility was that
Chinese ML directly triggered the cross-linking of CD95 death receptor, similar
to a mechanism of Con A-induced activation of the TCR. To investigate the
involvement of CD95 in ML-induced apoptosis, we used Jurkat-R T cell clones
that had been selected for resistance to CD95. In these cells, 60 kDa Chinese
ML induced apoptosis with dose-escalation and kinetics, indicating that CD95
was not required for 60 kDa Chinese ML-induced cytotoxicity. Apoptosis induced
by 60 kDa Chinese ML was CD95-independent.In summary, we show that Chinese ML is a potent immunomodulation
agent on human gd T cell cytotoxicity, apoptosis induction and the cytokine network.
These results might, therefore, provide promising insights and a molecular
rationale to determine the therapeutic efficacy and clinical benefit of Chinese
ML in the treatment of different human cancers. The further characterization
of the Chinese ML’s subunits is still going on.
Acknowledgement
We thank Mr. Ovid Da SILVA, Editor,
Research Support Office, Research Center, CHUM, University of Montreal
(Montreal, Canada), for his editorial assistance.
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