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Originally published online as doi:10.1189/jlb.0106056 on February 6, 2007

Published online before print February 6, 2007
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(Journal of Leukocyte Biology. 2007;81:1276-1286.)
© 2007 by Society for Leukocyte Biology

(S)-Armepavine inhibits human peripheral blood mononuclear cell activation by regulating Itk and PLC{gamma} activation in a PI-3K-dependent manner

Chih-Peng Liu*, Yuh-Chi Kuo{dagger},1, Chien-Chang Shen{ddagger},§, Ming-Hsi Wu*, Jyh-Fei Liao*, Yun-Lian Lin{ddagger},§, Chieh-Fu Chen*,|| and Wei-Jern Tsai*,{ddagger},§,1,2

* Institute of Pharmacology, National Yang-Ming University, Taipei, Republic of China;
{dagger} Institute of Life Science, Fu-Jen University, Taipei, Republic of China;
{ddagger} National Research Institute of Chinese Medicine, Taipei, Republic of China;
§ National Tai-Tung University, Taitung, Taiwan, Republic of China; and
|| Qing-Dao University, Qingdao, Republic of China

2 Correspondence: Room 517, Laboratory of Biochemistry, National Research Institute of Chinese Medicine, No. 155-1, Sec. 2, Li-Nung St., Shih-Pai, 112, Taipei, Taiwan, R.O.C. E-mail: wjtsai{at}nricm.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chinese herbs are useful edible and medicinal plants for their immune modulatory functions. We have proven that (S)-armepavine (C19H23O3N; MW313) from Nelumbo nucifera inhibits the proliferation of human PBMCs activated with PHA and improves autoimmune diseases in MRL/MpJ-lpr/lpr mice. In the present study, the pharmacological activities of (S)-armepavine were evaluated in PHA-activated PBMCs. The results showed that (S)-armepavine suppressed PHA-induced PBMC proliferation and genes expression of IL-2 and IFN-{gamma} without direct cytotoxicity. Inhibition of NF-AT and NF-{kappa}B activation suggested phospholipase C{gamma} (PLC{gamma})-mediated Ca2+ mobilization and protein kinase C activation were blocked by (S)-armepavine. Phosphorylation of PLC{gamma} is regulated by lymphocyte-specific kinase (Lck), ZAP-70, and IL-2-inducible T cell kinase (Itk). We found (S)-armepavine inhibited PHA-induced phosphorylation of Itk and PLC{gamma} efficiently but did not influence Lck or ZAP-70 phosphorylation. In addition, ZAP-70-mediated pathways, such as the association of linker for activation of T cells with PLC{gamma} and activation of ERK, were also intact in the presence of (S)-armepavine. Finally, reduction of phosphoinositide 3,4,5-trisphosphate formation and Akt phosphorylation suggested that (S)-armepavine inhibited Itk, and PLC{gamma} phosphorylation might be a result of the influence of PI-3K activation. Addition of exogenous IL-2 or PMA/A23187 rescued PBMC proliferation in the presence of (S)-armepavine. Therefore, we concluded that (S)-armepavine inhibited PHA-induced cell proliferation and cytokine production in a major way by blocking membrane-proximal effectors such as Itk and PLC{gamma} in a PI-3K-dependent manner.

Key Words: lymphocytes • proliferation • phytohemagglutinin • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue inflammation, a harmful immune response that produces tissue injury, is characteristic of serious diseases such as asthma, system lupus erythematosus (SLE), and rheumatoid arthritis (RA) [1 ]. There is now convincing evidence that IFN-{gamma} and IL-2, secreted by T cells, play an important role in lung inflammation and asthma [2 ]. In patients with RA, the levels of T cells and cytokines such as IL-2, IFN-{gamma}, and TNF-{alpha} have been shown to elevate significantly in rheumatoid synovium, suggesting a possible pathological role for T cells and cytokines [2 ]. The role of cytokines in the pathogenesis of T cell-mediated disease is modulating the activation of the lymphocyte population [3 ]. One of the therapeutic objectives in tissue inflammation is to reduce the local inflammatory response [4 ]. Blockade of the T cells activation, proliferation, and cytokine production is one of such anti-inflammatory means [5 ].

Interaction of T cells with antigens or PHA initiates a cascade of biochemical events and protein production, which induces resting T cells to enter the cell cycle, proliferation, and differentiation [6 ]. Following the TCR activation, ZAP-70 binds to the CD3 {zeta}-chain, resulting in the phosphorylation by lymphocyte-specific kinase (Lck) [7 ]. Activated ZAP-70 has been shown to phosphorylate linker for activation of T cells (LAT) to recruit phospholipase C{gamma} (PLC{gamma}) and then results in PLC{gamma} phosphorylation and activation [8 ]. Full activation of PLC{gamma} also depends on IL-2-inducible T cell kinase (Itk) [9 ], which is phosphorylated by Lck [10 ] in conjunction with PI-3K [11 ]. Activated PLC{gamma} triggers an increase in the concentration of intracellular calcium ([Ca2+]i) and activates various isoforms of protein kinase C (PKC) [12 ]. Downstream effectors of a [Ca2+]i increase and PKC activation are NF-AT and NF-{kappa}B, respectively [13 , 14 ]. These two important transcription factors are reported to regulate many cytokine gene expressions including IL-2 and IFN-{gamma} [15 ]. ERK activation affects c-fos expression, and ERK and c-fos are involved in T cell growth [16 ]. TCR couples signals to ERK via PKC-dependent [17 ] or LAT-growth factor receptor-binding protein 2-dependent pathways [18 ].

Although pathogenesis is not well known, aberrant immune responses with excessive and uncontrolled T cells are presented in autoimmune diseases such as SLE [19 ]. Lymphocytes are the primary targets for pharmacological therapy of SLE. Recently, the inhibition of TCR-mediated signaling was identified as one of the working mechanisms of many anti-inflammatory drugs. Some immunosuppressive agents such as corticosteroids and cyclosporin A are available to treat autoimmune diseases [20 ]. Although the mechanisms by which these agents exert their effects on SLE are not fully understood, their inhibitions against T lymphocytes are strongly implicated [21 ]. However, the uses of these agents are still accompanied by serious complications that limit their administration and overall clinical benefit [22 ]. Traditional Chinese herbs are now widely acknowledged for their immunomodulatory and antitumor activities [23 ]. Nelumbo nucifera is commonly applied in traditional Chinese medicine for treatment of diarrhea and tissue inflammation [24 , 25 ]. Recent studies also found that N. nucifera showed hepatoprotective and free radical scavenging effects [26 ]. The manner in which N. nucifera exerts its anti-inflammatory effects is not well understood, although it has been ascribed to its ability to block TNF-{alpha} and TGF-ß formation [27 ]. In our previous studies, we found that the ethanolic extracts of N. nucifera inhibited proliferation of human PBMCs activated by PHA [28 ]. (S)-Armepavine (C19H23O3N; MW313) is the major active principle of N. nucifera responsible for this effect [29 ]. The in vivo study indicates (S)-armepavine can inhibit T cell function to reduce cytokine and autoantibody production and finally improve diseased progression of SLE in MRL/MpJ-lpr/lpr mice [30 ]. To elucidate the pharmacological activities of (S)-armepavine further, we analyzed the effects of (S)-armepavine on cell proliferation, the protein production, and genes expression of IL-2 and IFN-{gamma}, activation of NF-AT and NF-{kappa}B, and membrane-proximal effectors such as PLC{gamma}, Itk, ZAP-70, Lck, and PI-3K in PBMCs stimulated with PHA to clarify its action mechanism in T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
(S)-Armepavine isolated from seeds of N. nucifera
The isolation of (S)-armepavine from seeds of N. nucifera was brief, according to the previous study [29 ]. Silica gel and Sephadex LH-20 column were used in this separation procedure. The structure of (S)-armepavine is shown (see Fig. 1A ).


Figure 1
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  		Figure 1. The structure of (S)-armepavine and its effects on PBMC proliferation and viability. (A) The structure of (S)-armepavine purified from N. nucifera. (B) PBMCs (2x105/well) were treated with an indicated concentration of (S)-armepavine, with or without PHA (5 µg/ml) for 3 days. The proliferation of cells was detected by [3H]thymidine uptake. After 16 h incubation, the cells were harvested, and then radioactivity was measured by liquid scintillation counting. **, P < 0.01, as compared with the vehicle and PHA-treated cells. (C) PBMCs (2x105 cells/test) were cultured with 100 µM (S)-armepavine for the indicated time and then supplemented with 10% alamarBlueTM for 3 h to determine the viability of PBMCs. Data are shown as mean ± SD of three independent experiments.

 
Preparation of PBMCs
Heparinized human peripheral blood was obtained from healthy volunteers. PBMCs were isolated by the Ficoll-Paque (specific gravity 1.077) gradient density method as described previously [23 ].

Lymphoproliferation test
The lymphoproliferation test followed the method described previously [23 ]. In brief, various concentrations of (S)-armepavine were applied to the cells (2x105/test), with or without 5 µg/ml PHA for 3 days. Subsequently, tritiated thymidine (3[H]thymidine; 1 µCi/well) was added. After 16 h incubation, the cells were harvested, and radioactivities in the filters were measured by liquid scintillation counting. The inhibitory activity of (S)-armepavine on PBMC proliferation was calculated.

Determination of cell viability
Approximately 2 x 105 cells/well PBMCs were cultured with vehicle (0.1% DMSO) or 100 µM (S)-armepavine for indicated times. Then, the cultures were supplemented with 10% alamarBlueTM reagent. After 3 h, the absorbance was measured at 570 nm and 600 nm, and the viability of PBMC treated with (S)-armepavine was calculated by the following formula: Viability (%) = experimental group (OD570 nm–OD600 nm)/control group (OD570 nm–OD600 nm) x 100.

Determination of cytokine production in PBMCs
PBMCs (2x105 cells/well) were cultured with varying concentrations of (S)-armepavine in combination, with or without PHA for 3 days. The cell supernatants were then collected and assayed for IL-2 and IFN-{gamma} concentrations with the enzyme immunoassays (EIA).

Extraction of total cellular RNA
The total cellular RNA was extracted from PBMCs by a method described previously [28 ]. Following stimulation, cells were lysed by RNA-BeeTM. Total cellular RNA was extracted with a phenol-chloroform mixture and precipitated with isopropanol. The concentrations of the extracted RNA were calculated by measuring the OD at 260 nm. The ratio of the OD at 260 nm:280 nm was always higher than 1.8.

Synthesis of the first-strand cDNA
Aliquots of 1 µg RNA were reverse-transcribed using the AdvantageTM RT-for-PCR kit according to the manufacturer’s instructions and the method described previously [28 ].

Real-time quantitative PCR
The real-time PCR was performed by the TaqMan PCR assay using an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA). The reaction conditions were 50°C for 2 min followed by 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. {Delta} Cycle of threshold ({Delta}CT) was calculated by subtracting the CT of GAPDH mRNA from the CT of IL-2 mRNA or IFN-{gamma} mRNA.

PCR
The PCR was performed in an air thermocycler, according to the manufacturer’s instructions, and followed the procedure as described previously [23 ]. Primers for c-fos (forward 5'-ATCAGCAGCATGGAGCTGAAGACC-3'; reverse 5'-CTGGGAACAATACACACTCCATGC-3', 607 bp) and GAPDH (forward 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'; reverse 5'-CATGTGGGCCATGAGGTCCACCAC-3', 983 bp) were designed from the published human cDNA sequence data. The amplified product was analyzed by agarose gel, and each band was quantitated with a laser-scanning densitometer (ImageMaster VDS, Pharmacia Biotech, Uppsala, Sweden).

Preparation of nuclear extraction
Cells (5x107/test) were treated with various concentrations of (S)-armepavine in the absence or presence of PHA for 15 or 60 min. The cells were suspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 0.1 mM PMSF) plus Nonidet P-40 (0.25%) for 15 min. After centrifugation, the nuclear pellet was resuspended in buffer B (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 2 µM leupeptin) for 30 min. The supernatants were collected, and the protein concentrations were measured by a Bio-Rad dendritic cell protein assay kit (Bio-Rad, Hercules, CA, USA).

Determination of NF-AT and NF-{kappa}B activation by EMSA
The gel-shift kits were used in binding reactions for EMSA, according to the manufacturer’s instructions. The sequences of NF-AT- and NF-{kappa}B-binding sites in the human IL-2 promoter were 5'-ACGCCCAAAGAGGAAAATTTGTTTCATACA-3' and 5'-AGTTGAGGGGACTTTCCCAGGC-3', respectively. In brief, 1 µg polydeoxy(inosinate-cytidylate) and 10 µg nuclear extracts were first incubated, and then 10 ng biotin-labeled probes were added, and the incubation continued for a further 30 min. For competition experiments, 100- to 200-fold excess unlabeled competitor oligos were added to the extracts before the addition of labeled probes. Samples were run on a 6% Tris/borate/EDTA (TBE) gel and transferred to a nylon, positively charged membrane. Following UV cross-linking, the membrane was incubated with streptavidin-HRP conjugate and detected with working substrate solution. Finally, the membrane was exposed to film for 1–10 min. Each band was quantitated by the laser-scanning densitometer.

Preparation of cytosolic and membrane extraction
PBMCs were suspended in lysis buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM Na3VO4, 0.2 mM PMSF, 50 µM NaF, 2 µM leupeptin, and 0.7 µg/ml pepstatin A) with 0.1% Triton X-100 for 10 min. The cytosolic fraction was collected by centrifugation at 10,000 g for 3 min. Pellets were resuspended in lysis buffer with 0.5% Triton X-100 for 60 min. The membrane fraction was collected from supernatant by centrifugation at 10,000 g for 20 min [23 , 31 ].

Preparation of immunoprecipitates
Total cellular proteins (100 µg) were incubated with the appropriate antibodies overnight at 4°C. Then, the supernatants were followed by incubation with protein G plus/protein A agarose for 2 h at 4°C. After washing three times, the immunoprecipitates were then subjected to immunoblotting.

Western blot analysis
The cell lysates were dissolved in the dissociation buffer (2% SDS, 1.5% 2-ME, 5% glycerol, 5% sucrose, and 0.002% bromophenol blue in 62.5 mM Tris-HCl, pH 6.8) and boiled for 10 min. Solubilized proteins were separated by 10% SDS-PAGE and transferred to polyvinylidine fluoride filters. After blocking for 1 h with 3% milk in TBST, the filters were incubated overnight at 4°C with proper dilutions of anti-PKC{theta}, anti-ZAP-70, antiphosphorylated (anti-p)-ZAP-70, anti-PLC{gamma}, anti-p-PLC{gamma}, anti-Itk, anti-LAT, anti-Lck, anti-ERK, anti-p-Akt, or anti-p-tyrosine (anti-p-Tyr) antibodies. The filters were washed and incubated for 1 h by appropriate secondary antibodies linked to HRP, and the stained bands were visualized by the ECL reagents.

[Ca2+]i measurement
Analysis of [Ca2+]i in PBMCs was modified from reports described previously [23 ]. The PBMCs were loaded with 1 mM fluo-3-acetoxymethyl (AM) at 37°C for 30 min and then resuspended in Ca2+-free medium to a density of 2 x 106 cells/ml. In each experiment, 1 ml PBMC suspension was equilibrated with an equal volume of 2 mM Ca2+-containing buffer at 37°C for 2 min. The suspension was added with vehicle (0.1% DMSO) or various concentrations of (S)-armepavine for 30 min and then stimulated with 5 µg/ml PHA at the 30th second. The fluorescence was measured by a fluorescence spectrophotometer (F-4500, Hitachi, Tokyo, Japan) with a multiwavelength time-scan program. The fluorescence activity was recorded at excitation wavelength of 505 nm and emission wavelength of 530 nm. The maximal ratio and minimal ratio were obtained with 20% digitonin and 50 mM EGTA, respectively.

Phosphoinositide (PI) mass assay
The extraction of PI was modified as the method described previously [32 ]. Cellular materials were precipitated by ice-cold 0.5 M TCA and washed with 5% TCA/1 mM EDTA. The acidic lipids were extracted in CHCl3:methanol:HCl (40:80:1, by volume) for 60 min, and the phases were then split by the addition of 0.75 ml CHCl3 and 1.35 ml 0.1 M HCl followed by centrifugation at 13,000 g for 5 min to separate the organic and aqueous phases. The organic phase was collected and dried down. The pellets were resuspended by the PI 3,4,5-trisphosphate (PIP3) assay buffer (50 mM HEPES, 150 mM NaCl, 1.5% Na cholate, pH 7.4) and sonicated in a water bath. The mass of PI was quantitated by a PIP3 Mass ELISA kit, according to the manufacturer’s instructions.

Materials
Reagents were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich Co. (St Louis, MO, USA). Anti-PKC{theta}, anti-ZAP-70, anti-PLC{gamma}, anti-p-PLC{gamma}, anti-LAT, anti-Lck, anti-p-Tyr, anti-integrin ß1, and anti-p-Akt antibodies were obtained from BD Biosciences (San Diego, CA, USA). Anti-p-ERK, anti-ERK, and anti-p-ZAP-70 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Itk antibody was obtained from Calbiochem (Darmstadt, Germany). PHA was purchased from Sigma-Aldrich. 3[H]Thymidine was purchased from New England Nuclear (Boston, MA, USA). alamarBlueTM reagent was obtained from BioSource International Inc. (Camarillo, CA, USA). The kits for EIA were from Diaclone Research (Stamford, CT, USA). RNA-BeeTM was purchased from Tel-Test (Friendswood, TX, USA). AdvantageTM RT-for-PCR kit was from Clontech (Palo Alto, CA, USA). PCR primers and TaqMan probes were purchased from Applied Biosystems. The gel-shift kit was from Panomics (Redwood City, CA, USA). Fluo-3-AM was obtained from Invitrogen (Carlsbad, CA, USA). Protein G plus/protein A agarose was from Merck. PIP3 Mass ELISA kit was purchased from Echelon Biosciences (Salt Lake City, UT, USA). The seeds of N. nucifera were purchased from a Chinese medicine shop in Taipei, Republic of China, and identified by Dr. Y-L. Lin.

Statistical analysis
Data were presented as mean ± SD, and the differences between groups were assessed with a one-way ANOVA test at a significant level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
(S)-Armepavine inhibited PBMC proliferation
To investigate the effect of (S)-armepavine on PBMC proliferation, PBMCs were treated with various concentrations of (S)-armepavine in the presence or absence of PHA (5 µg/ml). As shown in Figure 1B , cells were stimulated with PHA for 3 days, significantly enhancing proliferation ~26-fold (331±10.6 cpm vs. 8858±220 cpm), as reflected by the increase in 3[H]thymidine uptake. Treatment with the vehicle (0.1% DMSO) did not affect the resting or the stimulated state of 3[H]thymidine uptake. (S)-Armepavine suppressed the proliferation of PHA-activated PBMCs significantly in a concentration-dependent manner. The corresponding degrees of inhibition for 25, 50, and 100 µM were 61.7 ± 1.7%, 87.8 ± 4.6%, and 95.0 ± 4.1%, respectively, with an IC50 of 11.9 µM. In comparison with the vehicle-treated group, the viability of PBMCs was not decreased significantly following treatment with 100 µM (S)-armepavine for 72 h (84.7±9.8% vs. 97.4±7%; Fig. 1C ). It indicated that the inhibitory effects of (S)-armepavine on PBMC proliferation were not a result of a direct cytotoxicity.

(S)-Armepavine blocked IL-2 and IFN-{gamma} production and mRNA expression in PBMCs
To elucidate the effect of (S)-armepavine on cytokine production, PBMCs treated with various concentrations of (S)-armepavine were incubated with or without PHA for 3 days, and the production of IL-2 and IFN-{gamma} was determined by EIA. The production of these cytokines in activated PBMCs was suppressed significantly by (S)-armepavine in a concentration-dependent manner (Fig. 2A and 2B ). At 100 µM, the stimulated production of cytokines in activated PBMCs was blocked completely by (S)-armepavine, and cytokine concentrations were almost the same as those in resting cells.


Figure 2
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  		Figure 2. Effects of (S)-armepavine on IL-2 and IFN-{gamma} production in PBMCs, which were treated by 0, 25, 50, and 100 µM (S)-armepavine, with or without PHA (5 µg/ml) for 3 days. The supernatants of each test were collected, and (A) IL-2 and (B) IFN-{gamma} concentration was determined by EIA. Data are shown as mean ± SD of three independent experiments. **, P < 0.01, as compared with the control group in PHA-activated cells.

 
Total cellular RNA was extracted from PHA-activated PBMCs in the presence or absence of 25, 50, and 100 µM (S)-armepavine for 18 h and subjected to real-time PCR. As shown in Table 1 , vehicle did not show any effect on IL-2 and IFN-{gamma} mRNA expression in PBMCs. In comparison with the vehicle-treated group, the {Delta}CT values of IL-2 and IFN-{gamma} were decreased significantly by PHA (P<0.001). (S)-Armepavine concentration-dependently reversed the {Delta}CT values of IL-2 and IFN-{gamma} in activated PBMCs. Thus, impairments of (S)-armepavine on IL-2 and IFN-{gamma} production were related to inhibit IL-2 and IFN-{gamma} mRNA expression in PBMCs stimulated with PHA.


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  		Table 1. Effects of (S)-Armepavine on IL-2 and IFN-{gamma} Transcriptions in PBMCs Determined by Real-Time PCR

 
(S)-Armepavine suppressed NF-AT and NF-{kappa}B activation in PBMCs
We used EMSA to determine the effect of (S)-armepavine on two major transcription factors, NF-AT and NF-{kappa}B, induced by TCR signaling and involved in IL-2 and IFN-{gamma} gene regulation [15 ]. As shown in Figure 3A , the shift of NF-AT was induced significantly by PHA stimulation for 15 min (Lane 3), and excess of unlabeled probes decreased the formation of this binding shift (Lane 4). (S)-Armepavine concentration-dependently reduced NF-AT activation in PHA-treated PBMCs (Lanes 5–7). As shown in Figure 3B , the binding activity of NF-{kappa}B in PBMCs was increased significantly by PHA stimulation for 1 h (Lane 3) as compared with the vehicle-treated group (Lane 2). An increase in NF-{kappa}B activation was attenuated by (S)-armepavine in a concentration-dependent manner (Lanes 5–7). However, probes alone (Lane 1) did not show any binding shift in both figures. It demonstrated that (S)-armepavine impaired IL-2 and IFN-{gamma} gene expression, which might be related to its interruption on the activation of NF-AT and NF-{kappa}B in PBMCs induced by PHA.


Figure 3
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  		Figure 3. Effects of (S)-armepavine on the activation of NF-AT and NF-{kappa}B in PBMCs. The nuclear extracts were prepared from PBMCs, treated as described on the top of the figure and incubated with biotin-labeled, conserved (A) NF-AT- or (B) NF-{kappa}B-binding sequences. Samples were run on a 6% TBE gel. The results were detected with streptavidin-HRP conjugate and working substrate solution. Arrows indicate the NF-AT/DNA or NF-{kappa}B/DNA complex, and the relative value of each NF-AT/DNA or NF-{kappa}B/DNA complex normalized to that in Lane 1 is shown at the bottom of the figure. Results show a representative experiment of three.

 
(S)-Armepavine attenuated PLC{gamma} phosphorylaton and its downstream signaling events, including Ca2+ mobilization and PKC activation
To study whether (S)-armepavine inhibits PHA-stimulated [Ca2+]i mobilization, the [Ca2+]i change of PBMCs was detected by a fluorescence spectrophotometer. Here, we showed that PHA induced a comparable pattern of Ca2+ mobilization in PBMCs (Fig. 4A ). The increase of [Ca2+]i was no different between the vehicle-treated and medium-treated PBMCs (data not shown). In addition, (S)-armepavine could not cause Ca2+ mobilization in resting cells (data not shown). The [Ca2+]i in 0, 25, 50, and 100 µM (S)-armepavine-treated PBMCs induced by PHA was 144.8 ± 11.3 nM, 117.4 ± 10.0 nM, 65.1 ± 13.1 nM, and 0.5 ± 6.1 nM, respectively. The results suggested that (S)-armepavine might attenuate PHA-stimulated [Ca2+]i, which was related to its inhibition on NF-AT activation in PHA-treated PBMCs.


Figure 4
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  		Figure 4. Effects of (S)-armepavine on Ca2+ mobilization, PKC activation, and PLC{gamma} phosphorylation in PBMCs. (A) Fluo-3-loaded PBMCs (2x106 cells/ml) were incubated with 0, 25, 50, and 100 µM (S)-armepavine for 30 min. After equilibration with 2 mM Ca2+-containing buffer, PHA (5 µg/ml) was added at the 30th second. The changes of fluorescence with time were recorded by a F-4500 fluorescence spectrophotometer with a multiwavelength time-scan program. (B) PBMCs (1.5x107 cells) were treated with various concentrations of (S)-armepavine in the presence or absence of PHA for 30 min. The membrane fraction was extracted and subjected to 10% SDS-PAGE. The upper blots were stained with PKC{theta} antibody, and the lower blots were stained with integrin ß1 antibody as the loading control. The ratio of PKC{theta}:integrin ß1 was indicated at the bottom. (C) PBMCs (1.5x107 cells) were treated with or without various concentrations of (S)-armepavine for 30 min and then stimulated with PHA for 5 min. Cell lysates (100 µg) were precipitated with PLC{gamma} antibody (0.5 µg), and the immunoprecipitates were subjected to 10% SDS-PAGE. The upper blots were stained with p-PLC{gamma} antibody, and the lower blots were stained using PLC{gamma} antibody. The ratio of p-PLC{gamma}:PLC{gamma} was indicated at the bottom. Representative data from more than three independent experiments are shown.

 
To realize whether (S)-armepavine affected PKC activation, the membrane fractions were extracted to detect the PKC translocation. As shown in Figure 4B , membrane expression of PKC{theta} was induced by the treatment of PHA for 30 min (Lane 3). (S)-Armepavine suppressed PKC{theta} content significantly in the plasma membrane in a dose-dependent manner (Lanes 4–6). These results indicated that (S)-armepavine might inhibit PHA-induced NF-{kappa}B activation by decreasing PKC{theta} activation.

The inhibitions on [Ca2+]i and PKC implied that (S)-armepavine might affect the upstream event, PLC{gamma}, on both molecules. PLC{gamma} immunoprecipitates were resolved on 10% SDS-PAGE, and p-Tyr levels were detected by anti-p-PLC{gamma} antibody. The identity of PLC{gamma} levels was confirmed by reblotting with anti-PLC{gamma} mAb. As shown in Figure 4C , PHA treatment enhanced phosphorylation of PLC{gamma} in PBMCs within 5 min (Lane 2). Although (S)-armepavine alone had no effect (Lane 3), it inhibited PLC{gamma} phosphorylation concentration-dependently in PBMCs induced by PHA (Lanes 4–6). It suggested that inhibitory effects of (S)-armepavine on [Ca2+]i and PKC activation in PBMCs were a result of attenuate phosphorylation of PLC{gamma}.

(S)-Armepavine did not influence Lck activation and ZAP-70 phosphorylation
The Lck, ZAP-70, and Itk cooperative actions are essentially required to assemble signaling molecules and to finally phosphorylate PLC{gamma} [7 , 9 ]. As autophosphorylation in the activation loop of Lck is necessary for its kinase activity [33 ], we used immunoprecipitation and Western blot analysis to determine the effects of (S)-armepavine on Lck phosphorylation in PBMCs stimulated by PHA. The result showed that various concentrations of (S)-armepavine treatment did not inhibit the p-Tyr of Lck in PHA-activated PBMCs (Fig. 5A , top). The similar loading control of Lck was shown at the bottom (Fig. 5A) .


Figure 5
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  		Figure 5. Effects of (S)-armepavine on phosphorylation of Lck and ZAP-70 in PBMCs. (A) PBMCs (1.5x107 cells) were treated with or without various concentrations of (S)-armepavine for 30 min and then stimulated with PHA for 1 min. Cell lysates (100 µg) were precipitated with Lck antibody (1 µg), and the immunoprecipitates (IP) were subjected to 10% SDS-PAGE. The upper blots were stained with p-Tyr antibody, and the lower blots were stained using Lck antibody to show a similar amount of Lck presented in each lane. The ratio of p-Tyr:Lck was indicated at the bottom. (B) PBMCs (1x107 cells) were treated with medium (Lane 1), vehicle (Lane 2), PHA (Lane 3), vehicle + PHA (Lane 4), 100 µM (S)-armepavine (Lane 5), 25 µM (S)-armepavine + PHA (Lane 6), 50 µM (S)-armepavine + PHA (Lane 7), or 100 µM (S)-armepavine + PHA (Lane 8), respectively, for 3 min. The upper blots were stained with p-ZAP-70 antibody, and the lower blots were stained with ZAP-70 antibody to show the similar amounts of ZAP-70 presented in each lane. The ratio of p-ZAP-70:ZAP-70 was indicated at the bottom. Results show a representative experiment of more than three.

 
To further confirm the effect of (S)-armepavine on Lck activation, the phosphorylation status of in vivo substrate ZAP-70 was examined. As shown in Figure 5B , the p-Tyr of ZAP-70 was induced significantly by PHA stimulation for 3 min in PBMCs (Lane 3). In addition, phosphorylation of ZAP-70 was not affected by vehicle alone (Lane 2) or (S)-armapvine alone (Lane 5). Neither vehicle (Lane 4) nor various concentrations (25, 50, and 100 µM) of (S)-armepavine (Lanes 6–8) could abrogate PHA-induced p-Tyr of ZAP-70. These results suggested that (S)-armepavine did not inhibit Lck kinase activity and ZAP-70 phosphorylation.

(S)-Armepavine did not suppress the ZAP-70-mediated association of PLC{gamma} with LAT, ERK phosphorylation, and c-fos gene expression
According to the results of Figure 5B , ZAP-70 phosphorylation was not affected by (S)-armepavine. To confirm this, the association of PLC{gamma} with LAT, the substrate of ZAP-70, was determined by PBMC proteins immunoprecipitated with anti-LAT antibody or anti-PLC{gamma} antibody. As shown in Figure 6A , PBMCs treated with PHA for 5 min presented significant association of PLC{gamma} with LAT (Lane 2). Preincubation with (S)-armepavine did not show a significant effect on the association of PLC{gamma} with LAT in activated PBMCs (Lanes 3–5). These results proved that (S)-armepavine did not influence ZAP-70 phosphorylation or the ZAP-70-mediated association of PLC{gamma} with LAT.


Figure 6
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  		Figure 6. Effects of (S)-armepavine on the interaction of PLC{gamma} with LAT, phosphorylation of ERK, and expression of c-fos mRNA in PBMCs. (A) Cells (1.5x107 cells) were preincubated with (S)-armepavine (0, 25, 50, 100 µM) for 30 min and then activated with PHA for 4 min. The immunoprecipitates prepared by LAT (a) or PLC{gamma} (b) antibody were subjected to 10% SDS-PAGE and immunoblotted with PLC{gamma} and LAT antibody (upper panels in a and b), respectively. The ratio of PLC{gamma}:LAT or LAT:PLC{gamma} was presented at the bottom of a and b. (B) PBMCs (1x107 cells) were treated with medium (Lane 1), vehicle (Lane 2), PHA (Lane 3), vehicle + PHA (Lane 4), 100 µM (S)-armepavine (Lane 5), 25 µM (S)-armepavine + PHA (Lane 6), 50 µM (S)-armepavine + PHA (Lane 7), or 100 µM (S)-armepavine + PHA (Lane 8), respectively, for 30 min. The blot was stained with p-ERK or ERK antibody. The ratio of p-ERK:ERK was indicated at the bottom. (C) PBMCs were cultured with various concentrations of (S)-armepavine in the presence or absence of PHA for 30 min. The total cellular RNA was extracted from PBMCs, and the RT-PCR was done as described in Materials and Methods. Following the reaction, the amplified products were run on a 1.8% agarose gel. Each band was quantitated by a laser-scanning densitometer, and the ratio of c-fos:GAPDH was shown at the bottom. The representative data from more than three independent experiments are shown.

 
In T lymphocytes, ERK phosphorylation depends on ZAP-70 and LAT, and ERK activation ultimately controls the mRNA expression of c-fos. (S)-Armepavine did not affect ZAP-70 phosphorylation to imply that the activations of downstream ERK and c-fos were intact. As shown in Figure 6B and 6C , stimulation of PBMCs with PHA induced the phosphorylation of ERK and c-fos mRNA expression greatly. (S)-Armepavine (25, 50, and 100 µM) did not show any effect on ERK phosphorylation and c-fos mRNA expression in PHA-activated PBMCs. These results indicated that (S)-armepavine did not influence ZAP-70 activation and the downstream signals such as ERK and c-fos.

(S)-Armepavine suppressed PIP3 formation, the PI-3K-mediated Akt phosphorylation, and subsequent Itk phosphorylation
PLC{gamma} is a substrate of Itk and ZAP-70 [34 ]. According to the results above, (S)-armepavine did not inhibit Lck and ZAP-70 activation, given the possibility of (S)-armepavine to inhibit the PHA-induced activation of Itk in PBMCs. To verify this, we determined the effects of (S)-armepavine on Itk phosphorylation in activated PBMCs. As shown in Figure 7A , p-Tyr of Itk was increased within 4 min in PHA-activated PBMCs (Lane 2), whereas preincubation of PBMCs with (S)-armepavine (25, 50, and 100 µM) prior to stimulation reduced PHA-induced p-Tyr of Itk (Lanes 3–5) significantly. It suggested that (S)-armepavine inhibited PLC{gamma} phosphorylation in PBMCs by decreasing Itk phosphorylation.


Figure 7
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  		Figure 7. Effects of (S)-armepavine on PIP3 formation and Akt and Itk phosphorylation in PBMCs (1.5x107 cells), which were preincubated with (S)-armepavine (0, 25, 50, 100 µM) for 30 min and then activated with PHA for 3 min. The PIP3 formation was followed by PIP3 Mass ELISA kit and shown in Panel C. The phosphorylation of Itk (A) and Akt (B) was detected by immunoprecipitation and Western blotting, respectively. Immunoprecipitates and total cellular lysates were subjected to 10% SDS-PAGE. The blots were stained with p-Tyr or p-Akt antibody and detected by Itk or ERK antibody to show a similar amount presented in each lane. The ratios of p-Tyr:Itk and p-Akt:ERK are indicated at the bottom of Panels A and B. Results show a representative experiment of more than three.

 
PI-3K generates 3'-PIs, which subsequently recruit Itk to the cell membrane, and Itk is then phosphorylated by Lck [9 ]. To realize whether Itk phosphorylation, inhibited by (S)-armepavine, was a result of attenuate PI-3K activity, the formation of PIP3 and the phosphorylation of Akt were detected. The results of Figure 7B demonstrated that (S)-armepavine presented a concentration-dependent decrease in PIP3 formation induced by PHA. At 100 µM, (S)-armepavine showed similar inhibition activity to 20 µM Ly294002, a PI-3K inhibitor. To confirm this, the phosphorylation of Akt, an indicator of PI-3K activation [35 ], was analyzed by Western blotting. As shown in Figure 7C , PHA induced comparable levels of Akt phosphorylation (Lane 3). In comparison with the untreated group, (S)-armepavine concentration-dependently inhibited phosphorylation of Akt in PBMCs activated with PHA (Lanes 4–6). We concluded that (S)-armepavine inhibited Itk phosphorylation in a PI-3K-dependent manner.

Exogenous IL-2 and PMA/A23187 rescue the PBMC proliferation in the presence of (S)-armepavine
To elucidate the possibility that (S)-armepavine blocks PI-3K and IL-2 production and results in a decrease in PBMC proliferation, PMA (50 ng/ml)/A23187 (2 µM) or IL-2 (20 ng/ml) was added into PBMC cultures in the presence of (S)-armepavine (25 µM), and the cell proliferation was determined by the 3[H]thymidine uptake method. As shown in Figure 8 , the inhibitory activity of (S)-armepavine on PBMC proliferation was rescued by exogenous IL-2 and PMA/A23187. Furthermore, the PI-3K inhibitor Ly294002 could not affect activated PBMC proliferation by addition of PMA/A23187. It suggested that blocking of PI-3K signaling and impairment of IL-2 production were responsible for deficient proliferation of (S)-armepavine-treated PBMCs.


Figure 8
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  		Figure 8. Effects of IL-2 and PMA/A23187 on PBMC proliferation in the presence of (S)-armepavine. PHA-activated PBMCs (2x105/well) were treated by IL-2 (20 ng/ml) or PMA (50 ng/ml)/A23187 (2 µM) in the presence of (S)-armepavine (25 µM) for 3 days. The proliferation of cells was detected by [3H]thymidine uptake (1 µCi/well). After a 16-h incubation, the cells were harvested by an automatic harvester, and then radioactivity was measured by liquid scintillation counting. Each bar represents the mean ± SD of three independent experiments with PBMCs from different individuals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
(S)-Armepavine, an isoquinoline alkaloid, first isolated from Uvaria chamae, has no direct cytotoxic effect on normal cells such as thymocytes but possesses a cytotoxic activity on L929 fibroblast cells [36 ]. Although (S)-armepavine is a known compound, this is the first report of (S)-armepavine identified from seeds of N. nucifera to show the immunomodulatory action on PBMCs. The present study demonstrated that (S)-armepavine reduced PLC{gamma} activity by inhibiting Itk phosphorylation in a PI-3K-dependent manner. As a result of attenuate proximal signals of TCR in PHA-activated PBMCs, (S)-armepavine suppressed the downstream signals such as [Ca2+]i, PKC, NF-AT, and NF-{kappa}B pathways. Finally, (S)-armepavine blocking PBMC proliferation might be a result of impairing the mRNAs and protein expression of IL-2 and IFN-{gamma}. The growth-suppressive actions of (S)-armepavine were not explained by a drug-induced reduction in cell viability.

It has been demonstrated that a series of cytokines including IFN-{gamma}, IL-2, and IL-4 are contained in carefully controlled T cells passing through the cell cycle [37 , 38 ]. The present study showed that IL-2 and IFN-{gamma} in PHA-activated PBMCs were decreased by (S)-armepavine, and the impairments of IL-2 and IFN-{gamma} production were related to the decrease of their mRNA expression. In addition, the inhibitory activity of (S)-armepavine on PBMC proliferation was rescued by exogenous IL-2. It suggested that reduction of IL-2 production was responsible for deficient proliferation of (S)-armepavine-treated PBMCs. The IL-2 and IFN-{gamma} promoters contain several regulatory elements that can bind different transcription factors [15 ]. IL-2 and IFN-{gamma} gene expression in PBMCs stimulated with PHA could be blocked by NF-{kappa}B, NF-AT, and PI-3K inhibitors (unpublished data), and initially, we determined the effect of (S)-armepavine on NF-AT and NF-{kappa}B induced by TCR signaling. The data of EMSA demonstrated that (S)-armepavine decreased the NF-AT and NF-{kappa}B DNA-binding activities. NF-AT and NF-{kappa}B also play physiological roles in cells [13 , 14 ], and it explains why the vehicle (0.1% DMSO) alone has slightly inductive effects on NF-{kappa}B and NF-AT activation in resting PBMCs. As Ca2+ is critical for NF-AT activation [13 ], it is likely that the inhibitory effect of (S)-armepavine on PHA-induced NF-AT activation involves a modulation of [Ca2+]i. In addition to Ca2+, PKC{theta} can activate NF-{kappa}B and AP-1 in T cells [39 ]. It indicates that (S)-armepavine-inhibited NF-{kappa}B activation may be related to its attenuation in PKC activation. The present study indicated that (S)-armepavine had no ability to inhibit the c-fos mRNA transcriptions. However, the preliminary data demonstrated that c-jun mRNA, another component of AP-1, was inhibited by (S)-armepavine in PHA-activated PBMCs (data not shown). Whether (S)-armepavine inhibits AP-1 activation remains to be solved. We predicted that the main target of (S)-armepavine at least located on the upstream effectors of PKC activation and Ca2+ mobilization for regulation of NF-{kappa}B and NF-AT activation and decrease of IL-2 and IFN-{gamma} gene expressions.

It has also been reported that the ZAP-70-mediated, LAT-associated complex colocalizes PLC{gamma} with Itk, which in turn phosphorylates PLC{gamma} [40 ]. As Lck is responsible for phosphorylation of ZAP-70 and Itk [7 , 9 ], Lck seems to initialize TCR-signaling steps leading to PLC{gamma} activation. In our study, (S)-armepavine could not inhibit Lck-mediated ZAP-70 phosphorylation or ZAP-70-mediated association of LAT with PLC{gamma}. To confirm these results, we determined the effects of (S)-armepavine on ERK phosphorylation and c-fos mRNA expression, which depend on ZAP-70 and LAT activation. The results indicated that ERK phosphorylation and c-fos transcripts were intact in (S)-armepavine-treated cells. (S)-Armepavine, however, inhibited PHA-induced PLC{gamma} phosphorylation in a Lck-independent and ZAP-70-indepentent manner; therefore, it is likely that (S)-armepavine attenuates PLC{gamma} phosphorylation by down-regulating Itk activation. In addition to Lck, Itk in T cells is regulated by PI-3K [10 , 11 ]. Wortmannin, a PI-3K inhibitor, abolishes Itk-mediated IL-2 gene induction and Itk p-Tyr [11 ]. PI-3K provides 3'-PIs to recruit Itk and Akt to the cell membrane and to result in phosphorylation of Itk and Akt [38 ]. With inhibition of PIP3 formation and Akt phosphorylation and no influence on Lck phosphorylation, the present study suggested that (S)-armepavine reduced Itk phosphorylation in a PI-3K-dependent manner. Ligation of the CD3 and/or CD28 results in rapid activation of PI-3K [41 ]. However, some downstream signaling effectors of CD3 and/or CD28, such as ZAP-70 and ERK, were still intact in the presence of (S)-armepavine. It suggested that (S)-armepavine did not inhibit PI-3K by interrupting PHA engagement with CD3 and/or CD28. In addition, the pleckstrin-homology domain of PLC{gamma} recognition of 3'-PIs might provide a mean to localize PLC{gamma} to the plasma membrane [42 ]. It suggests activation of PLC{gamma} is also regulated by PI-3K.

The PI-3K/Akt pathway regulates lymphocyte survival through inhibition of proapoptotic Bad and induction of antiapoptotic Bcl-2 [38 ]. Although Akt plays an important role in cell survival [38 ], the present results indicated that there was no significant cell death in PBMCs after treatment with 100 µM (S)-armepavine for 3 days. The previous data also showed that (S)-armepavine had no cytotoxic effect on mouse splenocytes and thymocytes [30 , 36 ]. We suggest that under 100 µM and during this time-frame, the inhibitory effects of (S)-armepavine on PBMC were not through cytotoxic effects, although we cannot exclude the possibility that (S)-armepavine may have toxic effects on PBMCs following chronic treatment or at higher concentrations. Although the structure of (S)-armepavine was distinguishable from Ly294002, another PI-3K inhibitor, the data showed that 100 µM (S)-armepavine and 20 µM Ly294002 had similar potential to inhibit PI-3K and reduce PIP3 formation. Although activated with PMA and A23187, neither (S)-armepavine nor Ly294002 could affect PBMC proliferation. Conversely, armepavine induces cell death on a CCRF-CEM leukemia cell line through decreasing expression of Bcl-2 and increasing expression of caspase-3, which are related to PI-3K activation [43 ]. Recently, we also used a non-T cell line, rat mesangial cells, as target cells to analyze the effects of (S)-armepavine. The preliminary results indicated that (S)-armepavine could decrease Akt phosphorylation in rat mesangial cells induced by LPS. It suggests that (S)-armepavine could inhibit PI-3K activation in different kinds of cells. The evidences above provide a powerful possibility that (S)-armepavine modulates PI-3K function to suppress PBMC proliferation.

In summary, the present study demonstrated that the inhibitory mechanism of (S)-armepavine on PHA-activated PBMC proliferation mainly involved the inhibition of PI-3K activation and its downstream effectors PLC{gamma} and Itk. We suggest that (S)-armepavine extracted from N. nucifera may also have acted to reduce tissue inflammation, in part, by inhibiting T lymphocyte function. It is known that SLE is related to overexpression of T cell-mediated, inflammatory responses [44 ]. T cells produce heightened levels of specific cytokines such as IL-2 and IFN-{gamma}, which contribute directly to the aberrant immune responses that ultimately lead to dysregulate autoantibody production, inducing the release of tissue-damaging substances and initiating an inflammatory response in SLE [45 ]. PI-3K plays a pathogenic role in SLE [46 ], and PI-3K inhibition blocks glomerulonephritis and extends the lifespan in MRL/MpJ-lpr/lpr mice [47 ]. We also provide clear evidence to verify the effects of (S)-armepavine on MRL/MpJ-lpr/lpr mice [30 ]. (S)-Armepavine reduces splenocyte proliferation, IL-2 and IFN-{gamma} production, autoantibody production, and glomerular hypercellularity in MRL/MpJ-lpr/lpr mice by inhibiting T cell function [30 ]. It suggests that significant attenuation of disease procession of (S)-armepavine in MRL/MpJ-lpr/lpr mice may be related to interruption of PI-3K activation in T cells. The relative contributions of these activities in vivo and in vitro demonstrate that (S)-armepavine may be a potential immunomodulator for the management of T cell-mediated diseases such as SLE.


   ACKNOWLEDGEMENTS
 
This study was supported partially by grants from National Science Council, Republic of China (NSC92-2320-B-030-014; NSC93-2320-B-077-014). We thank Dr. Yeu Su for technical comments about EMSA and Dr. Young-Ji Shiao for the support of inhibitors to identify the action mechanism of (S)-armepavine.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received January 24, 2006; revised December 11, 2006; accepted January 1, 2007.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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