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Introduction
IFN-g is a Th1-type cytokine and is principally generated by T lymphocytes. Two pathways participate in
IFN-g expres-sion signaling: one is the IFN-g-directed signaling in which
IFN-g promotes signal transducer and activator of transcription1
(STAT1) expression and activation, further enhances T-box transcription factor (T-bet expression), leading to the
IFN-g expression. The other is the IL-12-directed signaling, in which IL-12 induces STAT4 expression and activation, serving to
amplify IFN-g production[1]. Interferon regulatory factor-1 (IRF-1) is a transcription factor induced by
IFN-g and regulates interferon-related genes.
IFN-g tightly regulates the expression of various chemokines, including macrophage inflammatory
protein (Mip)-1a, Mip-1b, regulated upon activation normally T-cell expressed and secreted (RANTES), inducible protein-10
(IP-10), monokine induced by IFN-g (Mig), and IFN-inducible T cell
a chemoattractant (I-TAC)[2]. The inhibition of these
chemokines' expression will be of therapeutic potential, especially in rheumatoid arthritis.
There are 3 major groups of mitogen-activated protein (MAP) kinases in mammalian cells: the extracellular
signal-regulated protein kinases (Erk), c-Jun N-terminal kinases (JNK)/stress-activated protein kinase (SAPK), and p38
mitogen-activated protein kinase (p38) MAP
kinases[3,4]. Erk is normally associated with proliferation and growth factor
induction. JNK and p38 are important for Th1
differentiation[5]. Both JNK and p38 MAP kinases are required for
IFN-g production by CD4+ Th1
cells[6].
(5R)-5-hydroxytriptolide (LLDT-8) is a novel triptolide derivate with potent immunosuppressive activities
in vivo. LLDT-8 prevented graft-versus-host disease and prolonged cardiac allograft
survival[7,8]. LLDT-8 inhibited type II
collagen-induced arthritis[9] and suppressed concanavalin A (ConA)-induced liver
injury[10]. Although LLDT-8 was demonstrated to
inhibit the IFN-g signaling in collagen-induced arthritis
mice[9], the immune responses in
vivo were too complex to clarify the precise action of LLDT-8. To better understand the underlying mechanisms of LLDT-8 action, in this study, we analyzed the
effect of LLDT-8 on IFN-g expression signaling in isolated murine T lymphocytes and on
IFN-g effector signaling in the macrophage cell line.
Materials and methods
Mice Male C57BL/6 mice and female BALB/c mice
(6_8 weeks old, 20_22 g) were purchased from Shanghai Experimental
Animal Center of Chinese Academy of Sciences (Shanghai, China; Certificate
No 99-003). The animals were housed under specific pathogen-free conditions. All experiments were carried out according to the National Institutes of Health Guide for
Care and Use of Laboratory Animals, and were approved by the Bioethics Committee of Shanghai Institute of Materia
Medica.
Preparation of LLDT-8 LLDT-8
(C20H24O7;
Mr=376; 99% purity) was synthesized from triptolide that was separated from
the Chinese traditional herb Tripterygium
wilfordii Hook F. The stock solution of LLDT-8 was prepared in
dimethyl-sulphoxide (DMSO) and further diluted with pathogen-free saline (for
in vivo study) or RPMI-1640 culture medium (for
in vitro study) supplemented with 10% fetal bovine serum (FBS), 100 kU/L penicillin and 100 mg/L streptomycin.
Preparation of splenocyte
Mice were sacrificed and their spleens were removed aseptically. A single cell suspension was
prepared and cell debris and clumps were removed. Erythrocytes were lysed with Tris-buffered ammonium chloride.
Mononuclear cells were washed and resuspended in culture
medium[11]. A B cell-depleted population was prepared by
immunomagnetic negative selection[12]. Briefly, cells were incubated with magnetic particles bound to goat anti-mouse Ig
(Qiagen, Valencia, CA, USA), followed by removing cell-bound magnetic particles with a rare earth magnet (Polysciences Inc,
Warrington, PA, USA). The purity of the T cells was analyzed with flow cytometry on a FACSCalibur (Becton Dickinson, San
Jose, CA, USA), and was consistently >90%.
Flow cytometry analysis of T cell activation marker expression
Anti-CD3 mAb (5 mg/L) were immobilized to a 24-well
plate and incubated for 2 h at 37 °C. The wells were washed twice with PBS, and B cell-depleted spleen cells
(3×109/L) from C57BL/6 mice were added in the presence of 100 nmol/L LLDT-8 for 24 h or 48 h. Cells were stained with fluorescein
isothiocyanate (FITC) conjugated anti-mouse CD25
(a chain) or CD69 mAbs, or phycoerythrin (PE) conjugated anti-mouse
CD154 mAb (BD Biosciences PharMingen, San Diego, CA, USA), and analyzed with flow cytometry.
Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cell division assay
B cell-depleted spleen cells from C57BL/6 mice were washed with PBS and suspended in PBS at
1×1010/L. CFSE (Molecular Probes, Eugene, OR, USA) was
then added to the cell suspensions at a final concentration of 1
mmol/L. The cells were incubated at 37 °C for 10 min and
washed 3 times with culture medium. The resulting CFSE-labeled cells
(3×109/L) were stimulated with ConA (5 mg/L) in the
absence or presence of 100 nmol/L LLDT-8 for 48 h. Then the cells were harvested and stained with PE-conjugated
anti-mouse CD4 or CD8 mAbs (PharMingen, USA), followed by flow cytometry analysis.
IFN-g production B cell-depleted spleen cells
(3×109/L) from C57BL/6 mice were cultured with 100 nmol/L LLDT-8 for 48
h in the anti-CD3 (5 mg/L)-coated plates. Culture supernatants were assayed for mouse
IFN-g by ELISA following the manufacturer's instructions (PharMingen, USA).
Ovalbumin (OVA) immunization assay
Female BALB/c mice were sc injected with 100 µg chicken egg OVA (Sigma, USA)
dissolved in PBS and emulsified with an equal volume of Freund's complete adjuvant containing the
Mycobacterium tuberculosis strain H37Rv (Wako Pure Chemical Industries Ltd, Osaka, Japan). The mice were intraperitoneally injected with
vehicle or 1 mg/kg LLDT-8 for 7 d.
Seven days later, B cell-depleted lymph node cells were prepared and cultured
(4×109/L) with OVA (100 mg/L) for 48 h.
Supernatants were collected for IFN-g assay. When cultured for 72 h, the cells were pulsed with 0.25 µCi
[3H]-thymidine for the last 8 h and harvested onto glass fiber filters. The incorporated radioactivity was counted using a Beta Scintillation
Counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA, USA).
RT-PCR and real-time PCR analysis
B cell-depleted spleen cells
(3×109/L) from C57BL/6 mice were cultured with 100
nmol/L LLDT-8 for 20 h in the anti-CD3 (5 mg/L)-coated plates. Cells were lysed using Trizol reagent (Invitrogen, Carlsbad,
CA, USA). Total RNA was isolated, reverse trans-cribed, and PCR amplified using specific primers. RT-PCR products were
visualized by electrophoresis through 1% agarose gels containing ethidium bromide. Relative quantita-tion with real-time
PCR was performed with (it seems no spell-out for SYBR) Green PCR Reagents (Qiagen, USA) and a continuous fluorescence
detection system (MJ Research, Waltham, MA, USA) were used according to the manufac-turer's instructions. The mRNA
levels were normalized to those of hypoxanthine-guanine phosphoribosyl-trans-ferase (HPRT).
Raw 264.7 cells were pretreated with LLDT-8 (400 and 800 nmol/L) for 2 h before stimulation with 50 kU/L
mIFN-g for
6 h, followed by RT-PCR analysis. LLDT-8 at 800 nmol/L did not affect cell
viability[13].
Western immunoblotting B cell-depleted spleen cells
(5×109/L) from C57BL/6 mice were pretreated with 100
nmol/L LLDT-8 for 2 h before stimulation with anti-CD3 (5 mg/L) plus anti-CD28 (2 mg/L) for 30 min. Cells were lysed in SDS sample buffer
(62.5 mmol/L Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.02% bromophenol blue) and boiled for 5
min at 100 °C. Aliquots were electrophoresed in a 12% polyacrylamide gel and transferred to nitrocellulose membranes
(Amersham Biosciences, Bucking-hamshire,UK). The membranes were treated with 10% non-fat milk for 1 h to block
nonspecific binding, rinsed, and (cell signaling Technology) incubated with a panel of rabbit polyclonal Abs against total Erk1/2,
phospho-Erk1/2, phospho-JNK/SAPK, phospho-p38 overnight at 4
°C. The membranes were then treated with horseradish
peroxidase (HRP)-conjugated anti-rabbit IgG for 1 h. Immune complexes were detected with a chemiluminescence substrate
(Pierce, Rockford, IL, USA) and exposed to Kodak X-ray film (Kodak, Rochester, NY, USA).
Statistical analysis Data are presented as mean±SEM or mean±SD where indicated. Student's
t-test and ANOVA were used to determine significance between the 2 groups where appropriate.
P<0.05 was considered significant.
Results
Effect of LLDT-8 on murine T lymphocyte immune
responses in vitro To assess the effect of LLDT-8 on T cell activation, B cell-depleted murine splenocytes were stimulated
with anti-CD3 and analyzed with flow cytometry. The expression levels of CD25, CD69, and CD154 indicated the activation
state of T cells. Compared with the control culture, LLDT-8 at 100 nmol/L reduced CD154 expression by 30%, but did not
affect CD25 and CD69 expressions in activated T cells (Figure 1A). Spleen cells were labeled with CFSE and activated with
ConA for 48 h to assess cell division of T cell subsets. Data were presented as Figure 1B. Both CD4 and CD8 T cells
underwent cell division after the ConA stimulation, which was markedly abrogated by 100 nmol/L
LLDT-8. Moreover, LLDT-8 at 100 nmol/L suppressed
IFN-g production with statistical significance in anti-CD3-stimulated T cells
(P<0.01; Figure 1C).
Treatment with LLDT-8 suppressed OVA-specific immune responses
To test the capacity of antigen-specific T cell
proliferation and IFN-g production after in vivo
LLDT-8 treatment, the mice were immunized with OVA and administered with
LLDT-8 at 1 mg/kg. The proliferation of lymph node (LN) cells was examined. The results in Figure 2A show that the LN cells
from the vehicle-treated mice proliferated well in response to OVA
ex vivo. LLDT-8 treatment greatly impaired the
OVA-specific proliferative response
(P<0.01). We investigated the capacity of LN cells to produce
IFN-g with OVA stimulation. B cell-depleted LN cells produced
large amounts of IFN-g in the mice treated with vehicle alone, whereas the increased production of
IFN-g was reduced in the LLDT-8-treated mice (Figure 2B).
Inhibition of IFN-g production signaling pathway by LLDT-8 treatment
in vitro We further elucidated the reduction of
IFN-g by LLDT-8 at the mRNA level. Data are summarized in Figure 3. The transcription for STAT1, T-bet, IL-12 receptor
b2 (IL-12Rb2), STAT4 and IRF-1 increased greatly in anti-CD3-activated T cells. In the presence of 100 nmol/L LLDT-8, their
mRNA expressions were retarded (Figure 3A). The T-bet, STAT4, and IRF-1 expressions were further
analyzed with real-time PCR. Similar inhibitory activity of
LLDT-8 was observed (Figure 3B).
Effect of LLDT-8 on phosphorylation of Erk1/2, SAPK/JNK and p38 in T cells
In mammalian species, MAP kinases are
involved in all aspects of immune responses, from the initiation phase of innate immunity to the activation of adaptive
immunity[5]. To better understand the inhibitory activity of LLDT-8, the activation of mitogen-activated protein kinase
(MAPK) was analyzed. Anti-CD3 plus anti-CD28 effectively induced the phosphorylation of Erk1/2, SAPK/JNK and p38
(Figure 4). We examined the effect of LLDT-8 on their phosphorylation levels at 30 min after stimulation
using Western immunoblot analysis. Treatment with 100 nmol/L LLDT-8 decreased the phosphorylation of these 3 kinases,
displaying the strongest inhibition on p-SAPK/JNK
(Figure 4).
LLDT-8 suppressed IFN-g-triggered chemokine expression
To evaluate the effect of LLDT-8 on IFN-g-triggered signaling,
expressions of a variety of chemokines were analyzed with RT-PCR at 6 h in
IFN-g-stimulated Raw 264.7 cells. Treatment with
IFN-g caused a dramatic increase in the transcripts of
Mip-1a, Mip-1b, RANTES, IP-10, I-TAC, and Mig, and this increase was
effectively abrogated by LLDT-8 (400 and 800 nmol/L; Figure 5).
Discussion
In our previous studies, LLDT-8 inhibited inflammatory and autoimmune diseases via suppressing T cell immune
responses[7,9]. Some of those beneficial effects of LLDT-8 seemed to be associated with the reduction of
IFN-g produc-tion. In this study, the effect of LLDT-8 on
IFN-g signaling was investigated.
LLDT-8 did not prevent T cells from activation. The expression of activation markers CD25 and CD69 was not affected by
LLDT-8. The CD154 (CD40 ligand) expression was only slightly reduced. Therefore, the inhibition of T cell immunity by
LLDT-8 might occur after the T cells were activated. LLDT-8 effectively prevented T cell division. Importantly, it seemed that
the inhibitory effect of LLDT-8 on CD4+ T cells was stronger than that on
CD8+ T cells. LLDT-8 suppressed the TCR-triggered
IFN-g production; the inhibitory activity of LLDT-8 in T cell proliferation and
IFN-g generation was further demonstrated in the OVA-immunized mice.
The IFN-g/STAT1/T-bet/IFN-g pathway and the
IL-12/IL-12Rb2/STAT4/IFN-g pathway mediate IFN-g
production[1]. LLDT-8 impaired the mRNA expressions of STAT1, T-bet,
IL-12Rb2 and STAT4, leading to the reduction of
IFN-g generation. IRF-1 is a transcription factor induced by
IFN-g. In the presence of LLDT-8, the IRF-1 expression was sup-pressed. In mammalian species, MAP kinases are involved in all aspects
of immune responses[5]. Erk activation is an important event of T cell activation. LLDT-8 reduced Erk1/2 phosphorylation,
indicating its role in T cell function. JNK and p38 MAP kinases have been implicated participating in
inflammation[4]. JNK2, 1 member of the JNK family, is critical for the initial production of
IFN-g during the differentiation of Th1
cells[14]. p38 MAP kinase pathway is crucial for T cell-mediated immunity and the development of Th1 responses. Activation of the p38 pathway
is required for IFN-g gene
transcription[5,15,16]. Moreover, persistent activation of p38 MAP kinase resulted in increased
IFN-g production by Th1 effector
cells[17]. The phosphorylation levels of SAPK/JNK and p38 were significantly reduced by
LLDT-8. This result indicated that LLDT-8 inhibited
IFN-g production and Th1 immune responses at least partially via preventing
JNK/SAPK and p38 activation.
IFN-g can regulate leukocyte trafficking through the induction of various chemokine genes, including
Mip-1a, Mip-1b, RANTES, IP-10, Mig, and
I-TAC[2]. IFN-g induces Mip-1a, Mip-1b, IP-10, and Mig in the presence of
STAT1[18]. IRF-1 response elements have been identified in the promoters of Mig, IP-10, and
I-TAC[19,20]. Thus, IFN-g may induce these
chemokine expressions also via the
IFN-g/STAT1/IRF-1 pathway. LLDT-8 effectively inhibited the transcription of these
chemokines in IFN-g-stimulated Raw 264.7 cells, implicating the inhibitory effect on
IFN-g-triggered immune responses.
In summary, LLDT-8 inhibited
IFN-g production via the blockade of
IFN-g/STAT1/T-bet/IFN-g signaling and
IL-12Rb2/STAT4/IFN-g signaling. LLDT-8 suppressed the
IFN-g-induced transcription of IRF-1 and chemokines. The blockade of
MAPK activation at least partially contributed to the suppressive effect of LLDT-8 on
IFN-g. Taken together, LLDT-8 was a promising
IFN-g inhibitor by targeting its production pathway and downstream effector pathway.
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