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The Journal of Immunology, 2008, 181, 1491-1498
Copyright © 2008 by The American Association of Immunologists, Inc.
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Novel Immunomodulatory Properties of Berbamine through Selective Down-Regulation of STAT4 and Action of IFN-{gamma} in Experimental Autoimmune Encephalomyelitis1

Yiping Ren2,*, Limin Lu2,*, Taylor B. Guo*, Ju Qiu*, Yiqing Yang*, Ailian Liu* and Jingwu Z. Zhang3,*,{dagger}

* Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiaotong University School of Medicine, and {dagger} E-Institutes of Shanghai Universities, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Berbamine (BM) is an herbal compound derived from Berberis vulgaris L commonly used in traditional Chinese medicine. In this study, we show that BM has potent anti-inflammatory properties through novel regulatory mechanisms, leading to reduced encephalitogenic T cell responses and amelioration of experimental autoimmune encephalomyelitis (EAE). The treatment effect of BM was attributable to its selective inhibitory effect on the production and action of IFN-{gamma} in CD4+ T cells, which was mediated through altered STAT4 expression in T cells. BM was found to up-regulate SLIM, a ubiquitin E3 ligase for STAT4, and promote STAT4 degradation, resulting in markedly decreased IFN-{gamma} production in CD4+ T cells in EAE mice. Regulation of IFN-{gamma} by BM had profound anti-inflammatory actions through its effect on both CD4+ T cells and APCs. BM-treated APCs exhibited reduced stimulatory function as a result of altered expression of PD-L1, CD80, and CD86 in treated mice. The treatment effect of BM in EAE was directly related to its action on IFN-{gamma}, and was abolished in IFN-{gamma} knockout mice. The study also confirmed that BM was able to inhibit NFAT translocation through effecting calcium mobilization in lymphocytes. However, this effect was not directly responsible for the treatment efficacy of BM in EAE. The study has important implications in our approaches to evaluating the utility of natural compounds in drug discovery and to probing the role of cytokine network in the development of autoimmune conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Both altered autoimmune T cell responses and dysfunction of the regulatory network of the immune system play an important role in human autoimmune pathologies, such as multiple sclerosis and rheumatoid arthritis (1, 2, 3). There is compelling evidence that these autoimmune diseases are characteristically associated with the over-production and activity of proinflammatory cytokines, most predominantly IFN-{gamma} and other Th1 cytokines, by autoreactive inflammatory T cells activated either systemically or at the site of inflammation (4, 5). Recently, the critical role of IL-17 in autoimmune inflammation adds an additional dimension to this complex network involving a battery of proinflammatory cytokines and the activity of related cross-talking signaling pathways (6, 7).

There has been growing interest to explore novel anti-inflammatory or immunomodulatory properties from herbal medicines. Chinese herbal medicine that has been practiced for thousands of years offers some unique advantages and provides a vast source of pharmaceutical material for the development of effective anti-inflammatory drugs. Natural compounds can be synthesized or purified from herbal medicines that have known indications for inflammatory disease conditions and often have a low toxicity profile (8, 9). The main obstacle, however, is that the mechanism of action of many of these natural/herbal compounds is largely unknown. In many cases, individual compounds in their natural herbal form, when singled out, are not highly effective. Finding effective anti-inflammatory natural compounds will not only enhance our understanding of regulation of the cytokine network and inflammatory cells and pathways involved, but also facilitate the identification of key chemical structures with anti-inflammatory properties for new drug design.

In this study, we investigated such an approach to identifying and characterizing novel anti-inflammatory compounds and the underlying regulatory mechanism using experimental autoimmune encephalomyelitis (EAE),4 a well-characterized animal model for multiple sclerosis. The herbal compound studied here was berbamine (BM), an alkaloid derived from Berberis vulgaris L that is commonly used in Chinese traditional medicine. Although its clinical indication as a single herbal component is not defined, studies mostly published in Chinese medical literature provide anecdotal indications of BM for arrhythmia and anti-inflammatory properties (10, 11). For example, BM exhibited an inhibitory effect on skin graft rejection (12) and delayed type hypersensitivity in mice (13). Its proposed property in the treatment of arrhythmia was thought to involve antagonism of calcium channel activity (14, 15). In this study, we evaluated potential regulatory properties of BM in the treatment of EAE. The goal of the study was to understand the underlying regulatory mechanism and specific interaction with various signaling molecules or pathways responsible for its treatment effect in EAE. The results presented in this study provide an important example in which anti-inflammatory herbal compounds can be studied to probe for novel signaling targets that are required for effective blocking of autoimmune T cell activation and functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Male C57BL/6 (B6) mice were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. IFN-{gamma} knockout mice (GKO) and IFN-{gamma} receptor knockout mice of the same B6 background were obtained from The Jackson Laboratory. Mice were maintained under pathogen-free conditions and genotyped before use.

Induction and treatment of EAE

Male B6 mice (6–8 wk) were immunized s.c. with a synthetic peptide (300 µg) of myelin oligodendrocyte glycoprotein (MOG residues 35–55). The sequence of the peptide was Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys and displayed a purity of >95% (GL Biochem). Immunization was performed by mixing MOG peptide in CFA containing 5 mg/ml heat-killed H37Ra, strain of Mycobacterium tuberculosis (Difco Laboratories). Pertussis toxin (200 ng; List Biological Laboratories) in PBS was administered i.v. on the day of immunization and 48 h later. For treatment of EAE, BM (Sigma-Aldrich) or DMSO (Sigma-Aldrich) as vehicle control was administered at 50 mg/kg i.p. daily from day 7 or day 10 postimmunization onwards. Mice were examined daily and scored for disease severity using the standard scale: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of one or two hind limbs); 3, paraplegia (complete paralysis of two hind limbs); 4, paraplegia with forelimb weakness or paralysis; 5, moribund or death. The animal protocol was approved by the institutional review board of the Institute of Health Sciences.

Histopathology

Spinal cords from mice transcardially perfused with 4% paraformaldehyde were dissected and postfixed overnight. Paraffin-embedded 5–10 µm spinal cord sections were stained with H&E or Luxol fast blue and then examined by light microscopy.

MOG-specific T cell response in vitro and ex vivo

For in vitro study, splenocytes isolated from EAE mice were cultured in triplicate in complete RPMI 1640 medium at a density of 5 x 105 per well in 96-well plates in the presence or absence of the MOG peptide (20 µg/ml). Either BM at the indicated concentrations or DMSO vehicle was added to the culture. For ex vivo study, splenocytes were isolated from BM- or vehicle-treated EAE mice and cultured in the presence or absence of the MOG peptide or purified protein derivatives of tuberculosis (Statens Serum Institut) at the indicated concentrations. To assess the function of APCs, splenocytes from BM- or vehicle-treated mice were re-stimulated with the MOG peptide (20 µg/ml) for 48 h. CD11b+ cells were then isolated by EasySep magnetic cell separation (StemCell Technologies) and cocultured with magnetically isolated CD4+ cells from EAE splenocytes (StemCell Technologies) at a ratio of 2:1 (16). All cultures were maintained at 37°C in 5% CO2 for 72 h. To measure T cell proliferation, cells were pulsed with 1 µCi [3H]thymidine during the last 16–18 h of culture before harvest. [3H]Thymidine incorporation was measured as cpm as detected by a MicroBeta β counter (PerkinElmer).

Measurement of cytokine production

Splenocytes (1 x 106 per well) isolated from BM-treated mice or vehicle controls were cultured in complete RPMI 1640 medium in the presence of the MOG peptide (20 µg/ml). Supernatants were collected after 48 or 72 h and diluted for measurement of IFN-{gamma}, TNF-{alpha}, IL-4, IL-5, IL-10, IL-13, IL-6, IL-17, and TGF-β by ELISA (R&D Systems) according to the manufacturer’s recommendations. A standard curve was performed for each plate and used to calculate the absolute concentrations of the indicated cytokines.

Immunoblotting and immunoprecipitation

Splenocytes from BM-treated or control mice were cultured in the absence or presence of the MOG peptide (20 µg/ml) for 24 h before cell lysis. Where indicated, a proteosome inhibitor, MG132 (Calbiochem), was used at 25 µg/ml. Nuclear protein extraction was prepared as described previously (17). Total cell or nuclear protein extracts were subjected to 6 or 10% SDS-PAGE. Immunoblot analysis was performed by transfer of proteins onto nitrocellulose membranes (Schleicher & Schuell Microscience) using a mini Trans-Blot apparatus (Bio-Rad). After 2 h of blocking, the membranes were incubated overnight at 4°C with specific primary Abs: anti-phosho-Tyk2, anti-phospho-Jak2, anti-Jak2, anti-phospho-STAT3, anti-STAT3, anti-phospho-STAT6, and anti-STAT6 (all obtained from Cell Signaling Technology), anti-Tyk2, anti-STAT4, anti-phospho-STAT1, and anti-STAT1 (all purchased from BD Biosciences), anti-phospho-STAT4 (Zymed Laboratories), anti-T-bet (Santa Cruz Biotechnology), anti-GATA3 (R&D Systems), anti-NFAT1 (Abcam), and anti-SLIM Ab, which was provided by Dr. M. J. Grusby (Department of Medicine, Harvard Medical School, Boston, MA). Anti-β-actin (Sigma-Aldrich) was used to detect β-actin as loading control, and anti-lamin B (Santa Cruz Biotechnology) and anti-GAPDH (KangChen Bio-tech) were used to detect lamin B and GAPDH as markers for nuclear and cytoplasmic fractions, respectively. After washing, subsequent incubation with appropriate HRP-conjugated secondary Abs for 1 h at room temperature, and extensive washing, signals were visualized by ECL (Pierce). For anti-NFAT1 immunoblotting, lymph node cells were cultured with BM, vehicle or cyclosporine, and stimulated with or without PMA and ionomycin (both obtained from Sigma-Aldrich) for 1 h. For detection of STAT4 ubiquitination, protein extracts were incubated overnight with anti-STAT4 Ab (R&D Systems) plus Protein G Sepharose 4 Fast Flow (GE Healthcare), and washed four times. Immunoprecipitated STAT4 was subjected to SDS-PAGE and immunoblotted with anti-ubiquitin Ab (Abcam).

Flow cytometry

For surface staining, splenocytes from BM-treated or control mice were re-stimulated with the MOG peptide (20 µg/ml) for 48 h. Expression of CD4, PD-1, ICOS, CD11b, PD-L1, CD80, and CD86 were analyzed by staining 0.5–1 x 106 cells with fluorochrome-conjugated specific mAbs or isotype controls (all purchased from BD Pharmingen) diluted in PBS containing 1% BSA according to the manufacturer’s protocol. For intracellular staining, CNS leukocytes were isolated from perfused mice as described (18). They were re-stimulated with 2 µg/ml ConA (Sigma-Aldrich) for 24 h, and GolgiPlug (BD Pharmingen) was added in the last 5 h (6). For staining of CTLA-4, IFN-{gamma} and IL-17 (all Abs purchased from BD Pharmingen), cells were permeabilized with the Cytofix/Cytoperm Plus kit (BD Pharmingen) and staining was performed according to the manufacturer’s protocol. Stained cells were washed and fixed with 1% paraformaldehyde and analyzed by a FACSAria cytometer (BD Biosciences).

For measurement of intracellular calcium concentration ([Ca2+]i), splenocytes from BM-treated or control mice were loaded with 4 µg/ml Fluo-4 (Molecular Probes) for 30 min at 37°C and washed twice with RPMI 1640 with 25 mM HEPES. Cells were incubated with BM or vehicle in the presence or absence of 10 µg/ml cytochalasin D (Sigma-Aldrich) or 1 mg/ml gadolinium (Sigma-Aldrich). Cells were warmed to 37°C, gated on lymphocytes, and analyzed for 20 s to establish baseline calcium levels. Then cells were stimulated with 1 µg/ml ionomycin, and calcium signals were acquired for an additional 300 s. Flow cytometric analysis was performed with FACSCalibur (BD Biosciences) using FlowJo software (TreeStar).

Statistics

Student’s t test was used to analyze the differences between the groups. Where appropriate, one-way ANOVA was performed initially to determine whether an overall statistically significant change existed before a two-tailed paired or unpaired Student’s t test was conducted. A value of p <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Treatment effect of BM on EAE and its regulatory properties on MOG-reactive T cell responses and cytokine production

The chemical form used in this study was berbamine dihydrochloride (Fig. 1). The compound had a low LD50 of 167 mg/kg when administered by i.p. injection in mice (19). As illustrated in Fig. 2A, when administered daily from day 7 postimmunization onwards, BM showed a significant inhibitory effect on the severity of EAE as compared with a vehicle control (DMSO). The clinical effect became overt at the time of disease onset (day 13 and day 14) and persisted over the course of EAE. Similar efficacy was observed when the treatment started from day 10 postimmunization (Fig. 2B). The observed clinical effect of BM was consistent with markedly reduced inflammation and demyelination in affected spinal cord lesions of treated mice as compared with those from untreated controls by histological analysis (Fig. 2C).


Figure 1
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  		FIGURE 1. Chemical structure of berbamine.

 

Figure 2
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  		FIGURE 2. Clinical course and severity of EAE in mice treated with BM. C57BL/6 mice were immunized with MOG35–55 peptide to induce EAE and were administered daily i.p. injections of BM (50 mg/kg, •) or vehicle control ({circ}) from day 7 (A) or 10 (B) postimmunization onwards. Mice were monitored and scored daily as described in Materials and Methods. Data are representative of three independent experiments. C, Histopathology of spinal cord tissue sections of EAE mice treated with BM or control by H&E staining (upper panels) and fast blue staining (lower panels), respectively.

 
The significant treatment effect of BM in EAE prompted us to investigate in detail potential regulatory mechanisms of the compound as to how it affected encephalitogenic T cell responses and to identify the target molecules through which BM might regulate autoimmune response. In our initial series of experiments, splenocytes were isolated from BM-treated and control EAE mice and characterized for ex vivo T cell reactivity and cytokine profile in response to antigenic challenge. The results revealed that BM altered MOG-reactive T cell responses in two ways. First, the proliferation of MOG-reactive T cells derived from BM-treated EAE mice was significantly inhibited compared with that of untreated controls in a dose-dependent manner (Fig. 3, A and B). This was not due to an adverse effect of BM on cell viability, as evidenced by propidium iodide-annexin V staining for cell death (data not shown). The effect of BM was not specific for MOG-reactive T cells because the T cell response to purified protein derivatives of tuberculosis was also inhibited (data not shown). Second, encephalitogenic T cells derived from BM-treated mice displayed a cytokine profile characterized by selective reduction of IFN-{gamma} in a dose-dependent manner (Fig. 3, C and D). The other anti- or pro-inflammatory cytokines commonly associated with EAE, including IL-17, were not markedly altered, whereas IL-4 production was significantly up-regulated (p < 0.05; Fig. 3C). The reduced production of IFN-{gamma} was also seen in CD4+ T cell infiltrates isolated ex vivo from brain and spinal cord tissue of BM-treated EAE mice, as compared with those of control mice by intracellular staining (7.46 ± 0.97% vs 16.34 ± 1.69%, p < 0.05), whereas the production of IL-17 was not affected (Fig. 3E).


Figure 3
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  		FIGURE 3. Encephalitogenic T cell response and cytokine profile in response to MOG peptide in EAE mice treated with BM. A, Encephalitogenic T cell response was measured as MOG-induced T cell proliferation in BM-treated (•) and control mice ({circ}). Splenocytes isolated 14 days postimmunization were stimulated with the indicated concentrations of the MOG peptide and examined for proliferation. Data are presented as mean tritiated thymidine incorporation (cpm ± SEM) in triplicates. B, MOG-reactive T cells isolated from EAE mice at 14 days postimmunization were cultured with the indicated concentrations of BM. Tritiated thymidine incorporation was measured at the end of the 72-h culture. C, Splenocytes from BM-treated mice (solid bars) or vehicle control (open bars) were challenged with the MOG peptide, and culture supernatants were collected at 48 h for cytokine measurement by ELISA. Data are presented as mean concentration (pg/ml ± SEM) of triplicate samples. D, MOG-reactive T cells were cultured with the MOG peptide and various concentrations of BM. Supernatants were collected for measurement of IFN-{gamma} production by ELISA. E, Mononuclear cells were isolated from spinal cords of BM-treated or control mice and stained for intracellular IFN-{gamma} and IL-17 in CD4+ cells. Data are representative of at least three independent experiments with similar results. Asterisks represent statistical significance between the groups (p < 0.05).

 
Selective down-regulation of IFN-{gamma} production by BM through increase in STAT4 degradation and up-regulation of its ubiquitin E3 ligase, SLIM

Detailed characterization was conducted to investigate the underlying mechanisms potentially responsible for regulatory properties of BM on IFN-{gamma}. We first examined the level of transcription factors associated with the differentiation of Th1 (STAT1 and T-bet) and Th2 (STAT6 and GATA3) by immunoblot analysis. Splenocytes from BM-treated or control mice were cultured in the presence or absence of the MOG peptide for 24 h. As shown in Fig. 4A, increased levels of STAT phosphorylation were induced by MOG stimulation. Notably, STAT1 phosphorylation, which was critically regulated by IFN-{gamma}, was markedly attenuated by BM treatment, whereas overall STAT1 level was not altered. Consistent with the shift of Th1 to Th2 induced by BM treatment was a trend for down-regulation of T-bet and up-regulation of STAT6 and GATA3.


Figure 4
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  		FIGURE 4. Down-regulation of IFN-{gamma} by BM through increased STAT4 degradation. Splenocytes from BM-treated or control mice were cultured in the absence or presence of the MOG peptide and analyzed by immunoblotting for phosphorylated and total STAT1 and STAT6, as well as T-bet and GATA3 (A), and phosphorylated and total Jak2, Tyk2, and STAT4 (B). C, MG132 was added during re-stimulation in the aforementioned culture, and STAT4 protein levels were examined by immunoblotting. D, Splenocyte lysates were immunoprecipitated (IP) with a STAT4 Ab followed by immunoblotting (IB) with a ubiquitin Ab. Total cell lysates were also immunoblotted with specific Abs as indicated. β-actin served as a loading control throughout the experiments. Data are representative of at least two independent experiments.

 
We further examined whether Jak/STAT signaling pathway was altered by BM treatment. Immunoblotting revealed no changes in STAT4 phosphorylation induced by MOG stimulation in BM-treated cells compared with the control (Fig. 4B), nor were there changes in the expression or phosphorylation of the upstream kinases Tyk2 and Jak2 (Fig. 4B). The level of protein expression as well as phosphorylation was not altered for IL-12R (data not shown). Interestingly, STAT4 protein level was markedly decreased (Fig. 4B). As STAT4 is subject to proteosome degradation (20), we hypothesized that BM treatment may enhance STAT4 ubiquitination and degradation. Our further study revealed that pretreatment with MG132, a commonly used proteosome inhibitor, significantly prevented BM-induced STAT4 reduction (Fig. 4C), indicating that BM treatment promoted STAT4 proteosomal degradation. Moreover, BM treatment significantly increased STAT4 polyubiquitination as evidenced by immunoblotting of immunoprecipitated STAT4 preparations from control and BM-treated cells (Fig. 4D). The increased STAT4 ubiquitination was associated with, in BM-treated cells, heightened expression of SLIM (Fig. 4D), which is identified as a ubiquitin E3 ligase for STAT4 (21).

Altered expression of costimulatory molecules and reduced stimulatory function of APC by BM treatment

We further addressed whether in addition to its characteristic effect on T cells, the regulatory properties of BM on MOG-reactive T cell responses were attributable in part to its ability to alter the function of APC. To this end, we examined the potential role of BM on the expression and function of selected costimulatory molecules, because IFN-{gamma} is known to regulate the activity of some of them (22, 23). Splenocytes were obtained from BM-treated or control EAE mice and analyzed ex vivo for the expression of CD28, ICOS, CTLA-4, PD-1, CD80, CD86, and PD-L1 (24). As shown in Fig. 5A, PD-L1 surface expression was significantly increased and that of CD80 and CD86 was decreased in CD11b+ cell populations of BM-treated EAE mice compared with those of control. Furthermore, the increased expression of PD-L1 in APC correlated with significantly reduced stimulatory function of BM-treated APC when cocultured with purified CD4+ MOG-reactive T cells (Fig. 5B). Expression of costimulatory molecules on the T cells was not different between BM treatment and control (data not shown).


Figure 5
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  		FIGURE 5. Effect of BM on CD11b+ APCs. A, Splenocytes from BM-treated or control mice were stained for PD-L1, CD80, and CD86 (open histograms) or isotype control (shaded histograms) after MOG stimulation and examined by flow cytometry in the CD11b+ cell population. B, CD11b+ cells isolated from the aforementioned cultures were cultured with CD4+ MOG-reactive T cells. Proliferation was measured as tritiated thymidine incorporation. Data are presented as mean cpm ± SEM of triplicates and are representative of at least three independent experiments with similar results.

 
BM inhibits ionomycin-induced calcium influx in MOG-reactive T cells without affecting intracellular calcium stores

There is anecdotal evidence suggesting that BM may have a property similar to a calcium channel blocker (14, 15). Such an effect would, in theory, affect activation-induced T cell proliferation in which calcium mobilization plays an essential role. We examined the fluorescent intensity of a Ca2+-sensitive fluorochrome as a readout of ionomycin-induced [Ca2+]i in MOG-reactive T cells pretreated with BM or control. As illustrated in Fig. 6A, BM treatment effectively blocked a sustained rise in [Ca2+]i induced by ionomycin in a dose-dependent manner. Our parallel experiments indicated that the observed effect was not related to the state of T cell activation, as activation markers including CD44, CD62L, CD25, and CD69 were not significantly altered in BM-treated cells (data not shown). Cytochalasin D is an F-actin disruptor that decouples endoplasmic reticulum-Ca2+ stores from extracellular Ca2+ influx through membrane channels (25). Further characterization showed that, in the presence of cytochalasin D, ionomycin-induced [Ca2+]i in cells treated with or without BM was significantly diminished compared with the positive control but was indistinguishable between them (Fig. 6B, left panel). Pretreatment of gadolinium, a blocker of plasma membrane calcium channels (26), prevented calcium influx in both BM-treated and control cells (Fig. 6B, right panel). These results suggest that BM inhibits activation-induced calcium influx without affecting the endoplasmic reticulum calcium stores.


Figure 6
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  		FIGURE 6. Effect of BM on calcium mobilization and NFAT nuclear translocation. A, Splenocytes from EAE mice were loaded with Fluo-4 and preincubated with 0.3 or 3.3 µg/ml BM or vehicle control. Baseline Fluo-4 fluorescence reflecting [Ca2+]i was determined and calcium influx was induced by 1 µg/ml ionomycin (Iono.) as indicated by an arrow. B, The effect of BM on calcium mobilization was studied in the presence or absence of cytochalasin D (CD, left) or gadolinium (Gd, right), respectively. C, Effect of BM on NFAT1 nuclear translocation was studied in nuclear lysates of PMA/ionomycin-stimulated cells (left) and unstimulated cells (right) by immunoblotting. Lymph node cells were cultured with BM, cyclosporine (positive control), or vehicle as indicated, with or without PMA/ionomycin stimulation. Nuclear and cytoplasmic extracts were immunoblotted with anti-NFAT1, anti-lamin B, or anti-GADPH Abs. Results are representative of two independent experiments.

 
Detailed characterization revealed that the observed anti-proliferative effect of BM was associated with its ability to inhibit nuclear translocation of transcription factor NFAT (27), as shown by markedly decreased NFAT1 in nuclear extracts of BM-treated T cells to levels comparable to those seen in unstimulated cells (Fig. 6C, left panel). Treatment with BM appeared to preferentially affect activated T cells as NFAT nuclear levels in resting T cells were not susceptible to the regulatory effect of BM under the same experimental conditions (Fig. 6C, right panel).

The treatment effect of BM in EAE is mediated through the action of IFN-{gamma}

Given the observed properties of BM in both IFN-{gamma} production and calcium channel activity in CD4+ T cells, it was critical to differentiate whether either or both effects contributed directly to the treatment activity of BM in EAE. To this end, EAE was induced in GKO mice of C57BL/6 background, and these mice were treated with BM or control using the same treatment protocol. The results showed that BM treatment had no significant effect on EAE in GKO mice (Fig. 7A), indicating that the treatment effect was mediated through IFN-{gamma}. Similar findings were confirmed in IFN-{gamma} receptor knockout mice (data not shown). Consistent with the lack of EAE treatment efficacy in GKO mice was the same level of heightened MOG reactivity exhibited by T cells from BM-treated and vehicle-treated GKO mice (Fig. 7A, inset). The expression and phosphorylation levels of STAT4 and STAT3, as well as the levels of T-bet, were analyzed. The results revealed that the levels of STAT4, and to a lesser extent T-bet, were reduced in encephalitogenic cells derived from BM-treated GKO mice compared with those from controls, whereas phosphorylated STAT4 and the levels of STAT3 were unaltered (data not shown). In addition, changes in the expression of PD-L1, CD80, and CD86 seen in APCs of BM-treated mice were not apparent in GKO mice (Fig. 7B). In contrast, the effect of BM on calcium mobilization was independent of IFN-{gamma}, as MOG-reactive T cells derived from GKO mice exhibited a pattern of decreased [Ca2+]i similar to that of wild-type mice (Fig. 7C).


Figure 7
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  		FIGURE 7. Lack of treatment effect by BM in IFN-{gamma} deficient mice. A, EAE was induced in GKO mice and treated with BM or vehicle control as described in Fig. 2A. Encephalitogenic T cell response in GKO mice treated with BM (solid symbol) or vehicle (open symbol) was measured by tritiated thymidine incorporation (inset). B, Flow cytometric analysis of the expression of PD-L1, CD80, and CD86 (open histograms) or isotype control (shaded histograms) in CD11b+ splenocytes from BM-treated or control GKO mice during peak of EAE. C, Effect of BM on ionomycin-induced calcium mobilization in both wild-type (WT) and GKO mice. Experiments were conducted as described in Fig. 6A. Data are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrated that BM, a natural alkaloid derived from Berberis, has unique anti-inflammatory properties and therapeutic potential for autoimmune inflammatory conditions. Our initial series of experiments determined that BM is effective in amelioration of EAE. It should be noted that BM treatment is equally effective in collagen-induced arthritis, an animal model for rheumatoid arthritis in our parallel experiments (data not shown), because both autoimmune conditions share many similarities involving the role of IFN-{gamma}. More importantly, the emphasis of the present study was to elucidate the novel regulatory mechanism induced by BM and to probe its specific interactions with molecular targets, which are responsible for its treatment effect in EAE. In this regard, we present detailed evidence that the treatment effect of BM is associated primarily with its selective inhibition of IFN-{gamma} through the Jak/Stat pathway in CD4+ T cells, including those of encephalitogenic potential. The observed effect is remarkably specific for IFN-{gamma}, a key player in T cell-mediated inflammation, and spares many other cytokines tested. The selective inhibition of IFN-{gamma} has profound effects on autoimmune response in EAE mice treated with BM. On one hand, the decreased production and action of IFN-{gamma} by BM treatment is found to induce shift of Th1 to Th2 in MOG-reactive CD4+ T cells as characterized by up-regulation of GATA3 and corresponding down-regulation of STAT1 and T-bet. On the other hand, in addition to its direct effect on CD4+ T cells, BM treatment appears to affect T cell function indirectly through altering the expression and function of costimulatory molecules on APC, such as PD-L1, CD80, and CD86, by the inhibition of IFN-{gamma}. The experiments in IFN-{gamma} knockout mice further confirmed that the observed regulatory effects were indeed attributable to the central role of IFN-{gamma} in BM-induced immune regulation. The data collectively provide a coherent model illustrating that these regulatory actions of BM work in concert to contribute to dampened autoreactive T cell responses in BM-treated EAE mice.

This unique property of BM seems to account for most, if not all, of the treatment effect because BM has no treatment effect in IFN-{gamma} or IFN-{gamma} receptor gene knockout mice. In line with this, although BM treatment also reduced STAT4 levels in encephalitogenic cells in GKO mice similar to that seen in the wild-type mice, the regulatory properties of BM could not be mediated further downstream to produce the treatment effect due to IFN-{gamma} deficiency. Our detailed characterization has provided new insights into the underlying molecular mechanism of action induced by BM. BM acts to up-regulate SLIM, a ubiquitin E3 ligase for STAT4 and to promote STAT4 proteosomal degradation without affecting the signaling that leads to STAT4 activation. It should be noted that STAT1 has also been reported to be a target of SLIM-mediated degradation (28). The observation that STAT1 protein level is unaltered by BM treatment suggests that BM is not a broad protein degradation enhancer but acts selectively toward STAT4 degradation. To our knowledge, BM is the first natural compound discovered so far to have such a selective regulatory property for IFN-{gamma}.

This study confirmed that BM treatment in EAE mice alters calcium channel activity in CD4+ T cells. This effect of BM is likely, in theory, to contribute to its ability to regulate T cell proliferation, which may account for the inhibitory effect of BM on the proliferation of encephalitogenic T cells in both ex vivo and in vitro settings. We demonstrated that BM treatment led to reduced NFAT nuclear translocation, resulting in decreased T cell proliferation through altered calcium influx. This regulatory mechanism related to calcium mobilization seems consistent with what has been proposed previously for anti-arrhythmic property of BM (10). However, our results fail to confirm that the effect of BM on calcium channel activity is responsible for the treatment effect. This is evident because BM treatment had no significant effect on the clinical course of EAE course or encephalitogenic T cell responses in GKO mice as opposed to those in wild-type mice. BM inhibition of calcium mobilization was also intact in T cells from GKO mice. The findings collectively indicate that the treatment effect of BM is dependent on its selective IFN-{gamma} antagonism and that it is not significantly associated with its effect on calcium channel activity.

The study sets an excellent example as to how understanding the underlying mechanism of action of a natural compound can provide critical insights into the role of identified target molecule(s) in an inflammatory cascade and their synergistic actions required for a compound to exert a treatment effect in a given disease. There are many good examples as to how novel biological or pharmacological mechanisms are identified through the analysis of a clinically efficacious natural/herbal compound. One of the recent examples is FTY720, which is derived from myriocin, a natural product isolated from the Chinese herb, Cordyceps sinensis. Based on its ability to sequester lymphocytes in the lymphoid organs, FTY720 was found clinically effective for graft rejection and other indications and is being tested for multiple sclerosis (29, 30, 31). Another such example is SM933, an artemisinin derivative, which was recently shown to possess anti-inflammatory and regulatory properties in EAE by selectively targeting NF-{kappa}B and the IFN-{alpha}/Rig-G/JAB-1 pathways (32). In the case presented here, identification of the molecular target of berbamine will go a long way toward novel effective therapies for human autoimmune and other inflammatory conditions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Natural Science Foundation of China (NSF-30430650 and NSF-30571731), the Shanghai Commission of Science and Technology (20014319207, 03DJ14009, 03XD14015, 04DZ19202, 04JC14040, and 04DZ14902), and the Shanghai Leading Academic Discipline Project (T0206). Back

2 Y.R. and L.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jingwu Zhang, Institute of Health Sciences, 225 South Chongqing Road, Shanghai 200025, China. E-mail address: jwzang{at}sibs.ac.cn Back

4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; BM, berbamine; GKO, IFN-{gamma} knockout; MOG, myelin oligodendrocyte glycoprotein; [Ca2+]i, intracellular calcium concentration. Back

Received for publication November 26, 2007. Accepted for publication May 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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