Andrographolide Attenuates Inflammation by Inhibition of NF- B

The Journal of Immunology

Andrographolide Attenuates Inflammation by Inhibition of NF-␬B Activation through Covalent Modification of Reduced Cysteine 62 of p501 Yi-Feng Xia,2* Bu-Qing Ye,2* Yi-Dan Li,2* Jian-Guo Wang,§ Xiang-Jiu He,† Xianfeng Lin,‡ Xinsheng Yao,† Dawei Ma,‡ Arne Slungaard,§ Robert P. Hebbel,§ Nigel S. Key,§ and Jian-Guo Geng3*§ NF-␬B is a central transcriptional factor and a pleiotropic regulator of many genes involved in immunological responses. During the screening of a plant extract library of traditional Chinese herbal medicines, we found that NF-␬B activity was potently inhibited by andrographolide (Andro), an abundant component of the plant Andrographis that has been commonly used as a folk remedy for alleviation of inflammatory disorders in Asia for millennia. Mechanistically, it formed a covalent adduct with reduced cysteine (62) of p50, thus blocking the binding of NF-␬B oligonucleotide to nuclear proteins. Andro suppressed the activation of NF-␬B in stimulated endothelial cells, which reduced the expression of cell adhesion molecule E-selectin and prevented E-selectinmediated leukocyte adhesion under flow. It also abrogated the cytokine- and endotoxin-induced peritoneal deposition of neutrophils, attenuated septic shock, and prevented allergic lung inflammation in vivo. Notably, it had no suppressive effect on I␬B␣ degradation, p50 and p65 nuclear translocation, or cell growth rates. Our results thus reveal a unique pharmacological mechanism of Andro’s protective anti-inflammatory actions. The Journal of Immunology, 2004, 173: 4207– 4217. he NF-␬B family of transcriptional factors regulates the expression of a wide spectrum of genes critically involved in host defense and inflammation. It has five cellular members: p105/p50 (NF-␬B1), p100/p52 (NF-␬B2), p65 (RelA), RelB, and c-Rel. All of them have a ⬃300-aa domain, which shares high homology, termed Rel homology domain, which mediates DNA binding, dimerization, and interactions with inhibitory cytoplasmic factors called I␬B proteins (1, 2). In almost all cell types, NF-␬B complexes are typically localized in the cytoplasm, where they bind to I␬B inhibitory proteins, including I␬B␣, I␬B␤, and I␬B⑀. Upon stimulation, I␬B proteins are rapidly phosphorylated by I-␬B kinases ␣ and ␤ (IKK␣ and -␤) and degraded via the ubiquitin-proteasome pathway. The degradation of I␬B proteins exposes the nuclear localization signal, resulting in nuclear shuttling of the p50/p65 heterodimer for the transcription of multiple targeting genes, such as those for cytokines (IL-1, IL-2, IL-6, IL-8, and TNF-␣), cell adhesion molecules (E-selectin, ICAM-1, and

T

*Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China; †Department of Phytochemistry, Shenyang Pharmaceutical University, Shenyang, Liaoning, China; ‡State Key Laboratory of Bioorganic & Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China; and §Vascular Biology Center and Division of Hematology, Oncology and Transplantation, University of Minnesota Medical School, Minneapolis, MN 55455 Received for publication October 31, 2003. Accepted for publication July 7, 2004. 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 Chinese Academy of Sciences (KSCX2-2-02), the National Science Foundation of China (39925015, 30130090, 30270649, 30370694, and 30340032), and the National Key Basic Research and Development Program (973 Program, 2002CB513000). 2

Y.-F.X., B.-Q.Y., and Y.-D.L. contributed equally to this study.

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Address correspondence and reprint requests to Dr. Jian-Guo Geng, Vascular Biology Center and Division of Hematology, Oncology and Transplantation, University of Minnesota Medical School, MMC 480, 14-152 Phillips Wangensteen Building, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail address: [email protected] Copyright © 2004 by The American Association of Immunologists, Inc.

VCAM-1), cyclooxygenase II, inducible NO synthase, immunoreceptors, hemopoietic growth factors, growth factor receptors, and several cell survival genes (3, 4). Activation of the NF-␬B pathway is currently known to be essential for the de novo synthesis of high levels of E-selectin (CD62E) mRNA and protein, a member of the selectin family of cell adhesion molecules (5). Upon challenges with cytokines TNF-␣, IL-1␤, and bacterial LPS, the endothelial cells of the capillary and venular vessels de novo synthesize and express E-selectin, which reacts with leukocyte carbohydrate ligands bearing the tetrasaccharide sialyl Lewisx and its derivatives as its minimal recognition motif. This interaction mediates initial attachment and rolling of neutrophils, monocytes, certain subsets of T lymphocytes, and eosinophils for recruitment of leukocytes to the site of infection or tissue injury (6). Herbal plants have been widely used as folk medicines in Asia for ⬎2 millennia. Many of them are still quite popular even today. For example, the plant Andrographis (Andrographis paniculate), which is a rich source for andrographolide (Andro)4 (7–9), has long been used as a folk remedy for alleviation of inflammatory disorders in China, India, Japan, and Korea. It currently is a prescribed medicine for treatment of laryngitis, diarrhea, and rheumatoid arthritis in China. However, little is known about the pharmacological mechanism of its known anti-inflammatory actions (10 –15). In this study we sought to identify, characterize, and test small m.w. antagonists for NF-␬B activation from traditional Chinese herbal medicines, such as Andro, for attenuation of inflammation. Our results demonstrate that Andro covalently conjugates

4 Abbreviations used in this paper: Andro, andrographolide; BAL, bronchoalveolar lavage; BCS, bovine calf serum; E-selectin-luc, an E-selectin promoter luciferase reporter plasmid; F5M, fluorescein-5-maleimide; 4H-Andro, 4-hydro-andrographolide; TFA, trifluoroacetic acid; vWF, von Willebrand factor; wt, wild type; Act1a, NF-␬B activator 1.

0022-1767/04/$02.00

Andro INHIBITS NF-␬B ACTIVATION

4208 reduced cysteine 62 of p50, thus preventing NF-␬B oligonucleotide binding to p50, inhibiting nuclear NF-␬B transcriptional activity, and attenuating inflammation in various in vitro assays and in vivo models.

Materials and Methods Reagents Andro was purchased from Aldrich (Milwaukee, WI), and 5 mg/ml (15 mM) stock was prepared by dissolving Andro in DMSO (Sigma-Aldrich, St. Louis, MO). 4-hydro-andrographolide (4H-Andro) was prepared via the palladium on activated carbon-catalyzed hydrogenation. Biotinylated deoxy-Andro was synthesized by the direct condensation of biotin with Andro under the assistance of 1,3-dicyclohexylcarbodiimide/1-hydroxybenzotriazole hydrate, whereas deoxy-Andro was prepared by the same reaction in the absence of biotin (16). Recombinant human TNF-␣ and IL-1␤ were purchased from Pierce Endogen (Shanghai, China). LPS (serotype 055:B5) was purchased from Sigma-Aldrich.

Cell culture Human cell lines of transformed embryonic kidney 293 cells (293; CRL1573) and promyeloid cells (HL-60; CCL-240) and a mouse cell line of fibroblast cells (NIH3T3; CRL-1658) were purchased from American Tissue Culture Collection (Manassas, VA). They were maintained in high glucose DMEM (for 293 cells and NIH-3T3 cells) and RPMI 1640 medium (for HL-60 cells) supplemented with 10% heat-inactivated FBS, 4 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin at 37°C in the presence of 5% CO2. HUVECs (within three passages) were prepared and cultured as previously described (17).

Transfection and luciferase assay 293 cells were transfected using calcium phosphate coprecipitation protocol as described previously (18). After transfection, the cells were lysed at 44 h, and the lysates were assayed for luciferase activities using the Luciferase Assay System (Promega, Madison, WI) (19, 20). Notably, the pCMV␤ vector (BD Clontech, Palo Alto, CA) was cotransfected so that the transfection efficiencies could be normalized with the ␤-galactosidase activities determined using a Luminescent ␤-Gal Detection Kit II (BD Clontech). For screening of the herbal extract library, 293 cells were cotransfected with plasmids of E-selectin-luc (1 ␮g), ␤-galactosidase (0.3 ␮g), and NF-␬B activator 1 (Act1) (1 ␮g). Herbal extracts (10 ␮g/ml) were added 24 h later for 12 h, followed by measurements of the luciferase and ␤-galactosidase activities. For inhibition experiments, the transfected cells were incubated with Andro for 12 h unless specifically indicated, before treatment with 500 U/ml TNF-␣.

EMSA 293 cells and HUVECs (within three passages) were treated with 500 U/ml TNF-␣, 750 U/ml IL-1␤, or 1.5 ␮g/ml LPS for 2 h before the nuclear protein preparations (19 –21). For in vivo inhibition, Andro was incubated for 12 h before induction with cytokines and bacterial endotoxins. For in vitro inhibition, Andro was incubated with the isolated nuclear proteins at 37°C for 30 min. In addition, the nuclear extracts from whole lung tissues of mice were prepared 1 h after LPS challenge. The wild-type p50 (aa 36 –385) and p50 C62S mutant plasmids were provided by Dr. D. Pe´ rezSala (22). The p50 C119S mutant was constructed to pET30a vector (Novagen, Madison, WI) using a MutantBEST Kit (TaKaRa, Dalian, China). They were expressed in Escherichia coli as hexahistidine fusion proteins and purified as described previously (22). The consensus oligonucleotide probes for NF-␬B (5⬘-AGT TGA GGG GAC TTT CCC AGG C-3⬘) and specificity protein 1 (5⬘-ATT CGA TCG GGG CGG GGC GAG C-3⬘) were purchased from Promega. The oligonucleotides were end-labeled with [␥-32P]ATP (Amersham Biosciences, Shanghai, China) and T4 polynucleotide kinase. The nuclear extracts (4 ␮g) or isolated recombinant p50 protein (100 ng) were added to a total 9-␮l reaction mixture of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 50 ␮g/ml poly(dI-dC)䡠poly(dI-dC). The 32Plabeled oligonucleotide probes (20 fmol) were added to each of the reaction mixtures and incubated at 22°C for 20 min. Protein-DNA complexes were separated from free DNA probes through 4% nondenaturing polyacrylamide gels in 0.5⫻ Tris-boric acid-EDTA buffer. Electrophoresis was performed at 15 V/cm. The gels were dried, and autoradiographies were analyzed using Band Leader 3.0 software (Magnitec, Tel-Aviv, Israel) (19 –21).

Pulldown assay and immunoblotting An aliquot of wild-type (wt) p50, p50 C62S, or C119S mutant (all at 1 ␮g) was precipitated with 2.5 ␮g of biotinylated deoxy-Andro at 37°C for 30 min, followed by 50 ␮l of ImmunoPure immobilized streptavidin beads (Pierce, Rockford, IL) at 4°C for 1 h to pulldown the proteins. To examine the binding activity of biotinylated deoxy-Andro to reduced and oxidized p50, p50 was treated with 10 mM 2-ME or 10 mM diamide, followed by dialyzing against PBS. The reducing state of p50 was verified by blotting with fluorescein-5-maleimide (F5M; Molecular Probes, Shanghai, China) (23). After washing five times with the lysis buffer, the beads were boiled in the presence of the SDS sample buffer. The proteins were separated on 10% SDS-polyacrylamide gels, transferred to Immobilon-P membranes (Millipore, Billerica, MA), and probed with a goat anti-p50 Ab (sc-1190; Santa Cruz Biotechnology, Santa Cruz, CA). In addition, p50 and p65 in the nuclear extracts and I␬B␣ in the cytosolic fractions of 293 cells were analyzed by Abs to p50, p65, or I␬B␣ (Imgenex, San Diego, CA), respectively.

HPLC purification and mass spectrometric analysis The p50 (0.5 mg) in PBS containing 1 mM EDTA, 5% glycerol (v/v), 1 ␮M 2-ME, and 0.01% Nonidet P-40 (v/v) was incubated with 250 ␮g of Andro at 37°C for 2 h. The reactant was injected into a ␮RPC C2/C18 column (Smart System; Amersham Biosciences), which had been equilibrated with 0.1% trifluoroacetic acid (TFA) in H2O. The protein was eluted with a gradient of 0 –100% 0.1% TFA in absolute acetonitrile at 240 ␮l/min for 45 min. The samples were dried out along with 0.5 ␮l of sinapinic acid (10 mg/ml) matrix in water/acetonitrile (a 1/1 dilution) containing 0.1% TFA. The MALDI-TOF mass spectrometric analysis was performed using a Voyager-DE PRO instrument (Applied Biosystems, Foster City, CA), operating in a linear mode. Calibration was performed externally using the standards from Sequazyme Mass Standards Kit (Applied Biosystems).

Flow cytometric assay HUVECs were treated with 500 U/ml TNF-␣, 750 U/ml IL-1␤, or 1.5 ␮g/ml LPS for 5 h and then detached by PBS containing 0.02% EDTA (Versene; Invitrogen Life Technologies, Gaithersburg, MD). After washing once, they were resuspended in PBS containing 0.5% BSA (PBS/BSA) and incubated with 3 ␮g/ml BBA2 (R&D Systems, Minneapolis, MN), followed by 15 ␮g/ml FITC-conjugated anti-mouse IgG Ab at 22°C for 1 h with end-to-end rotation. For inhibition experiments, HUVECs were preincubated with DMSO (a solvent control), 15 ␮M Andro, or 4H-Andro for 12 h before treatment with cytokines and endotoxin. Cells were spun down (1500 rpm for 5 min), and supernatants were discarded. Each aliquot was then resuspended in 0.5 ml of PBS/BCS for immediate flow cytometric analysis (FACScan; BD Biosciences, Mountain View, CA).

Laminar flow assay HUVECs were seeded on the polystyrene slides precoated with 1% gelatin, and the confluent monolayers were used in these experiments. Slides were fitted into a parallel plate laminar flow chamber as described previously (24). HL-60 cells were resuspended at 0.5 ⫻ 106 cells/ml in PBS supplemented with 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA. They were injected through the flow chamber at 22°C using a syringe pump. The wall shear stress was 2 dyne/cm2. The numbers of bound cells were quantified by videotape recordings of 10 –20 fields of view obtained (3– 4 min after flowing cells through the chamber) while scanning the lower plate of the flow chamber using a ⫻10 objective lens. For inhibition experiments, HUVECs were preincubated with 10 ␮g/ml mouse IgG1 or BBA2 (an E-selectin blocking IgG1 mAb) at 22°C for 20 min. Alternatively, HUVECs were treated with 15 ␮M Andro or 4H-Andro for 12 h before stimulation with cytokines and bacterial endotoxins.

Acute peritonitis model An aliquot of Andro or 4H-Andro (5 ␮g/g mouse body weight) was injected i.p. After 12 h, 400 U/g TNF-␣ or 500 U/g IL-1␤, in the presence or the absence of Andro or 4H-Andro (5 ␮g/g mouse body weight), was given i.p. Alternatively, the same dose of Andro or 4H-Andro was given 2 h after cytokine challenge. The mice were killed 4 h later (25, 26). For LPS challenge, an aliquot of Andro or 4H-Andro (5 ␮g/g mouse body weight) was injected i.p. on day 0, followed by the i.p. administration of LPS (1 ␮g/g mouse body weight) with or without Andro or 4H-Andro (5 ␮g/g mouse body weight) on days 1 and 2. Alternatively, the same dose of Andro or 4H-Andro was given i.p. 2 h after the LPS challenge. The mice were killed on day 3 (27). The assay of mouse acute peritonitis was performed as previously described (28).

The Journal of Immunology Endotoxin shock model BALB/c mice (7 wk old) were i.p. injected once with saline, Andro, and 4H-Andro (5 ␮g/g mouse body weight). Each mouse was i.p. injected with 0.75 mg of LPS in saline 2 h later. Alternatively, the same dose of Andro or 4H-Andro was given i.p. 2 h after the LPS challenge. The survival rates were monitored continuously for 7 days (27).

Allergic lung inflammation mode Mice were sensitized and challenged essentially as previously described (29). In brief, mice were sensitized with 20 ␮g of OVA (Sigma-Aldrich) and 2 mg of alum i.p. on days 0 and 5. Sham-immunized mice received alum alone. On days 11, 12, 13, and 14, DMSO, Andro, or 4H-Andro (5 ␮g/g mouse body weight) was given i.p. twice. On days 12, 13, and 14, mice were challenged by an aerosol of 1% OVA in PBS twice (separated for 4 h) for 1 h. Bronchial airway lavage (BAL) was performed at 24 h after the aerosol challenge. The BAL cells were stained with Wright’s staining buffer, and cell differentials were enumerated based on morphology and staining profile. Immunohistochemistry analysis of E-selectin, VCAM-1, and von Willebrand factor (vWF) in fixed and embedded mouse lungs was conducted as described previously (19, 30). The positive vWF-staining blood vessels were used as the total number of vessels in the tissues for calculating the ratios of the positive E-selectin- and VCAM-1-staining vessels.

Cell proliferation assay 293 cells, NIH-3T3 cells, and HUVECs (5 ⫻ 103 cells/ml) were plated into 96-well tissue culture plates, and the indicated amounts of Andro or 4HAndro were added 5 h later. They were then cultured for 48 h, and the cell growth rates were measured (30).

4209 induced luciferase activities. These data are consistent with the findings from the Act1-activated NF-␬B screening assay (Fig. 1B). Andro inhibits the binding of NF-␬B oligonucleotide We investigated whether Andro could prevent the binding of NF-␬B oligonucleotide to nuclear proteins. The 32P-labeled NF-␬B oligonucleotide did not bind to nuclear proteins before stimulation of 293 cells with TNF-␣ (Fig. 2A). In contrast, it bound avidly to nuclear proteins after TNF-␣ treatment. Andro, but not its structural analog 4H-Andro, attenuated the binding of NF-␬B oligonucleotide in a dose-dependent manner (IC50 ⫽ ⬃15 ␮M). As the specificity control, Andro apparently had no detectable effect on the binding of 32P-labeled specificity protein 1 oligonucleotide to nuclear proteins (Fig. 2B). To verify the above findings, we also examined whether Andro could directly abrogate the binding of NF-␬B oligonucleotide to the isolated nuclear proteins. Indeed, the NF-␬B probe bound avidly to the nuclear proteins isolated from the TNF-␣-activated 293 cells (Fig. 2C). Pretreatment of the nuclear proteins with Andro interfered with this binding in a dose-dependent manner (IC50 ⫽ ⬃15 ␮M). Interestingly, preincubation with DTT, a reducing reagent, markedly prevented the inhibitory effect of Andro. Apparently, Andro inhibits NF-␬B activation by blocking the binding of NF-␬B oligonucleotide to nuclear proteins, possibly through interaction with a cysteine residue(s) of NF-␬B protein(s).

Results

Screening for NF-␬B antagonists

Andro binds to reduced cysteine 62 of p50

The activation of NF-␬B pathway is currently known to be essential for de novo synthesis of high levels of E-selectin (CD62E) mRNA and protein (5). In a search for NF-␬B antagonists, we used the E-selectin promoter luciferase reporter assay and screened a plant extract library isolated from several hundred medicinal herbal plants commonly used in treating inflammatory and cardiovascular disorders as well as for cancers in China. During this screening, we identified only one purified compound, called Andro, which inhibited NF-␬B activation. The chemical structures of Andro, a bicyclic diterpenoid lactone, and its derivatives, 4H-Andro (an inactive analog), deoxy-Andro (an active analog generated during the chemical reaction of biotin coupling), and biotinylated deoxy-Andro (used for precipitation experiments) are presented on Fig. 1A. Transfection of 293 cells with an E-selectin promoter luciferase gene reporter for NF-␬B activation had minimal luciferase activity (Fig. 1B, first bar), whereas cotransfection with Act1 plasmid (an endogenous activator of NF-␬B) resulted in ⬎10-fold higher luciferase activities (Fig. 1B, second bar). To confirm the assay specificity, we cotransfected 293 cells with the I␬B␣ S32/ 36A mutant plasmid, which prevents proper ubiquitinization due to mutation of the two key phosphorylation sites, leading to specific inhibition of NF-␬B activation. As expected, the I␬B␣ S32/36A mutant plasmid completely abolished the luciferase activity of Eselectin promoter (Fig. 1B, third bar), attesting to the NF-␬B dependency of the assay. Notably, preincubation of the transfected 293 cells with Andro inhibited luciferase activity in a dose-dependent manner, demonstrating its inhibitory activity for NF-␬B activation. We then examined whether Andro could inhibit NF-␬B activation induced by TNF-␣. Treatment with 15 ␮M Andro potently inhibited the luciferase activities induced by TNF-␣ in a timedependent manner (Fig. 1C). Also, the 12-h treatment with Andro clearly reduced luciferase activities triggered by TNF-␣ in a dosedependent manner (IC50 ⫽ ⬃10 ␮M; Fig. 1D). The structural analog 4H-Andro was much less potent in inhibition of the TNF-␣-

Consistent with the above observations, the NF-␬B probe bound robustly to purified recombinant p50 protein after its dialysis in a buffer containing 0.1 mM 2-ME. Preincubation with Andro decreased the binding of NF-␬B oligonucleotide to p50 and p50 C119S mutant in a dose-dependent manner (IC50 ⫽ ⬃10 ␮M; Fig. 3A). In contrast, Andro failed to inhibit the binding of NF-␬B probe to p50 C62S mutant. Pretreatment of p50 with DTT completely prevented the inhibitory effects of Andro on the binding of NF-␬B oligonucleotide to p50. However, post-treatment of p50 with DTT was incapable of neutralizing the inhibitory effect of Andro on this interaction (Fig. 3B). To corroborate the above findings, we tried to detect the direct binding of Andro (a molecular mass of 350) to p50 using mass spectroscopic analysis. The spectrum of p50 showed a peak of m/z ⫽ 40,561, which corresponded to the calculated molecular mass of p50, together with a peak of m/z ⫽ 20,272 (doubly charged; Fig. 3C). The spectrum of Andro-treated p50 had peaks of m/z ⫽ 40,881 and m/z ⫽ 20,438 (double charged; Fig. 3D), which was fully compatible with the formation of a covalent adduct between one molecule of p50 and one molecule of Andro, with concomitantly forming one H2O molecule (a molecular mass of 18) during this chemical reaction. To gain insight into the biochemical mechanism, we explored whether biotinylated deoxy-Andro, which was generated during the chemical reaction of direct condensation of biotin with Andro, could directly react with p50. Deoxy-Andro was as active as Andro for inhibition of NF-␬B activation (data not shown). Biotinylated deoxy-Andro avidly precipitated wt p50 and p50 C119S mutant, but barely precipitated p50 C62S mutant (Fig. 3E). Furthermore, purified recombinant p50 protein was treated with DTT or diamide to generate reduced or oxidized p50, respectively. F5M, a thiol-modifying reagent, bound preferentially to the DTTtreated p50 over the diamide-treated p50, confirming the successful reduction and oxidation of p50 by these treatments (Fig. 3F). In parallel experiments, biotinylated deoxy-Andro precipitated more

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FIGURE 1. Andro inhibits NF-␬B activation. A, The structures of Andro, 4H-Andro, deoxy-Andro, and biotinylated deoxy-Andro. B, 293 cells, cotransfected with E-selectin-luc, and Act1 plasmid, were treated with Andro and assayed for luciferase activity. C, The transfectants were treated with 15 ␮M Andro at the indicated times before TNF-␣ stimulation. The results are the mean ⫾ SD from triplicate measurements of three separate experiments. D, The transfectants were treated with the indicated amounts of Andro or 4H-Andro for 12 h before TNF-␣ stimulation. The results are the mean ⫾ SD from triplicate measurements of three separate experiments.

reduced p50 than oxidized p50. The identical result was also obtained when the same membrane was reblotted with streptavidinconjugated HRP (data not shown). Taken together, our findings demonstrate that Andro reacts covalently with reduced cysteine 62 of p50. Andro suppresses E-selectin expression and attenuates leukocyte adhesion to activated endothelial cells As NF-␬B activation is critical for de novo synthesis of E-selectin, an inducible cell adhesion molecule for leukocytes, on vascular

endothelial cells (5, 6), we examined whether Andro, by inhibition of NF-␬B activation, could down-regulate the expression of Eselectin, leading to the reduction of leukocyte adhesion to stimulated endothelial cells. The NF-␬B oligonucleotide did not appreciably bind to nuclear proteins extracted from resting HUVECs (Fig. 4A). In contrast, it bound avidly after treatment with TNF-␣, IL-1␤, or LPS, respectively. Andro, but not 4H-Andro, attenuated the binding of NF-␬B oligonucleotide to the nuclear proteins extracted from the activated HUVECs. BBA2, a leukocyte adhesionblocking mAb to human E-selectin, bound to the TNF-␣, IL-1␤, or


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FIGURE 2. Andro inhibits the binding of NF-␬B oligonucleotide to nuclear proteins. A and B, The TNF-␣-stimulated 293 cells were treated with Andro and 4H-Andro, followed by the incubation of the nuclear proteins with labeled oligonucleotides. The results are the mean ⫾ SD values of three separate experiments. C, The nuclear proteins, without or with DTT, were isolated from the stimulated 293 cells before the incubation with Andro. The results are the mean ⫾ SD of three separate experiments.

LPS stimulated HUVECs, but not to resting HUVECs (Fig. 4B). Compared with 4H-Andro, Andro abolished the binding of BBA2 to the activated HUVECs. Using a laminar flow assay that mimics the hemodynamic status of capillary venules, we examined the effects of Andro on adhesion of human promyeloid HL-60 cells to stimulated endothelial cells. HL-60 cells adhered minimally to the resting HUVECs at a shear stress of 2.0 dyne/cm2, but they adhered avidly to HUVECs after stimulation with TNF-␣, IL-1␤, or LPS (Fig. 4C). BBA2, but not preimmune isotype-matched mouse IgG1, inhibited this adhesion, indicating that the observed adhesion of HL-60 cells to the activated HUVECs was mainly mediated by E-selectin. As expected, Andro, but not 4H-Andro, markedly inhibited the adhesion of

HL-60 cells to the stimulated HUVECs. These data indicate that Andro can inhibit the cytokine and bacterial endotoxin-induced overexpression of E-selectin and thus reduce the adhesion of leukocytes to the activated HUVECs. Andro reduces peritoneal infiltration of neutrophils, prevents endotoxin shock, and attenuates allergic lung inflammation We further evaluated the anti-inflammatory activity of Andro in the mouse models of acute peritonitis, endotoxin shock, and allergic lung inflammation. We proposed that if activation of NF-␬B were essential in the pathogeneses of these in vivo models of inflammation, blocking NF-␬B activation by Andro could dramatically attenuate these inflammatory lesions. We first examined the

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FIGURE 3. Andro reacts covalently with p50. A, The labeled NF-␬B oligonucleotide bound to p50 and its C119S, C62S mutant. However, preincubation of Andro prevented binding of the NF-␬B probe to p50 and its C119S mutant, but not to its C62S mutant. The results are the mean ⫾ SD values of three separate experiments. B, Preincubation with DTT blocked this binding, whereas postincubation with DTT failed to block this interaction. The results are the mean ⫾ SD values of three separate experiments. C and D, Mass spectrometric analysis of p50 without or with Andro preincubation. E, Biotinylated deoxy-Andro pulled-down p50 and the C119S p50 mutant, but not the C62S p50 mutant, detectable to the anti-p50 Ab. F, Biotinylated deoxy-Andro precipitated more reduced p50, but less oxidized p50, detectable to F5M and to the anti-p50 Ab.


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FIGURE 4. Andro suppresses NF-␬B activation of the stimulated endothelial cells and inhibits the peritoneal infiltration of neutrophils. A, The cytokineand LPS-stimulated HUVECs were treated with Andro and 4H-Andro, followed by EMSA. The results are the mean ⫾ SD of three separate experiments. B, The expression of E-selectin on the stimulated HUVECs was determined by BBA2 (a leukocyte adhesion blocking mAb to E-selectin). Results were presented as histograms of the log fluorescence intensities from 104 cells from one representative of three independent experiments. C, Adhesion of human promyeloid HL-60 to HUVECs was measured under flow in the absence (⫺) or the presence (⫹) of TNF-␣, IL-1␤, or LPS. For Ab inhibition, the stimulated HUVECs were preincubated with mouse preimmune IgG or BBA2. For compound inhibition, HUVECs were pretreated with Andro or 4H-Andro before stimulation with TNF-␣, IL-1␤, or LPS. D and E, Acute peritonitis was induced by i.p. injection of mice with saline (n ⫽ 3–5), TNF-␣, IL-1␤, or LPS (model; n ⫽ 9 –11), Andro (n ⫽ 8 –11), and 4H-Andro (n ⫽ 7–9). Andro and 4H-Andro were given either before (D) or after (E) cytokine or LPS challenge. The results are the mean ⫾ SD values of single measurement of multiple mice. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.

effects of Andro in the mouse model of acute peritonitis. Compared with saline treatment, the i.p. injection of TNF-␣, IL-1␤, and LPS induced peritoneal infiltration of leukocytes (the model; Fig. 4D). Before TNF-␣, IL-1␤, or LPS challenge, administration of Andro, but not 4H-Andro, could significantly reduce the peritoneal deposition of leukocytes ( p ⬍ 0.01). To mimic the clinical setting, we also examined whether treatment of Andro after cytokine and LPS challenges could reduce inflammation in this model. Indeed, administration of Andro 1 h after treatment with TNF-␣, IL-1␤, or LPS

inhibited the peritoneal deposition of neutrophils (Fig. 4E), even though the inhibitory effects became less dramatic ( p ⬍ 0.05). In addition, we investigated the effects of Andro in the murine model of septic shock. As shown in Fig. 5A, the i.p. injection of 0.75 mg of LPS/mouse caused 50% of the mice (six of 12 testing mice) to die from endotoxin shock. Administration of Andro, but not 4H-Andro, rescued all mice from the death in this animal model of endotoxin shock. Again, administration of Andro 2 h after LPS treatment also reduced the mortality rate

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FIGURE 5. Andro prevents septic shock and attenuates allergic lung inflammation. A and B, Mice were i.p. injected with saline (model), Andro, and 4H-Andro (5 ␮g/g mouse body weight) either before (A) or after (B) LPS challenge, and the survival rates were monitored continuously. C, EMSA of NF-␬B oligonucleotide binding to the nuclear proteins extracted from the mouse lungs after treatment with saline, Andro, and 4H-Andro before LPS challenge. Sham, no LPS; model, LPS; Andro, Andro and LPS; 4H-Andro, 4H-Andro and LPS. D, Leukocyte counts and differentiations in BAL from the mice without (sham) or with OVA aerosol challenge (model), which were pretreated with Andro (Andro) or 4H-Andro (4H-Andro). E, Leukocyte counts and differentiations in blood from the mice without (sham) or with OVA aerosol challenge (model), which were pretreated with Andro (Andro) or 4H-Andro (4H-Andro). F, Expression of E-selectin and VCAM-1 on the endothelial cells of blood vessels from the mouse lungs without (sham) or with OVA aerosol challenge (model), which were pretreated with Andro (Andro) or 4H-Andro (4H-Andro).

(75%, or three of 12 testing mice; Fig. 5B). To directly demonstrate the involvement of NF-␬B activation in this endotoxin shock model, the nuclear proteins extracted from whole mouse lungs were subjected to EMSA. Compared with saline (the sham group), LPS (the model group) clearly activated NF-␬B.

Andro, but not 4H-Andro, decreased the binding of NF-␬B oligonucleotide to the nuclear proteins extracted from the mouse lungs (Fig. 5C). It should be pointed out that based on the data from the supershift experiments using Abs to p50 or p65, the faster migrating (lower) band in the lung extracts from the

The Journal of Immunology model group was mainly p50/p50 homodimer, whereas the slower migrating (higher) band was mainly p50/p65 heterodimer (data not shown). Furthermore, we explored the pharmacological actions of Andro in the mouse model of OVA-induced allergic lung inflammation. As shown in Fig. 5D, aerosol challenge of mice with OVA clearly triggered the infiltration of leukocytes, especially eosinophils, in BAL. The i.p. administration of Andro, but not 4H-Andro, potently reduced the accumulation of leukocytes (including eosinophils) in BAL (Fig. 5D), but not in blood (Fig. 5E), ruling out the possibility of hemopoietic inhibition as the potential mechanism for reduction of leukocytes in BAL of Andro-treated mice. Consistently, the strong expression of E-selectin and VCAM-1 was detected in the lung tissues from the model group compared with those in the lung tissues from the sham (saline) group (Fig. 5F). Treatment with Andro, but not with 4H-Andro, significantly reduced the staining of E-selectin and VCAM-1. Together, our results not only confirm the critical roles of NF-␬B activation in these inflammatory models, but also demonstrate the efficacy of Andro in treating various types of inflammatory responses in vivo. Andro fails to inhibit I␬B␣ degradation, p50 and p65 nuclear translocation, and cell growth As the degradation of I␬B proteins and the nuclear translocation of p50 and p65 could modulate NF-␬B activity, we investigated the roles of Andro on these biochemical and cellular processes. Using an Ab to I␬B␣, we detected I␬B␣ protein in untreated 293 cells (Fig. 6A). TNF-␣ caused a rapid degradation of I␬B␣ in a timedependent manner. Andro apparently did not attenuate the degradation of I␬B␣. Immunoblotting of the extracted nuclear proteins with the Abs to p50 and p65 detected p50 and p65 in the TNF-␣activated 293 cells, but not in the resting 293 cells (Fig. 6B). Pretreatment with Andro had no detectable effects on the nuclear translocation of p50 and p65. Furthermore, Andro had no inhibitory actions on the growth rates of 293 cells, NIH-3T3 cells, and HUVECs (Fig. 6C). These results suggest that Andro apparently

4215 does not interfere with the degradation of I␬B␣, p50, or p65 and has no apparent cytotoxicity to 293 cells, NIH-3T3 cells, and HUVECs in vitro.

Discussion Our study has identified the molecular mechanism of Andro’s antiinflammatory actions, i.e., it acts as a small m.w. antagonist for NF-␬B activation by covalent modifying reduced cysteine 62 of p50. The dramatic reduction of the peritoneal deposition of neutrophils induced by cytokines and LPS (Fig. 4, D and E), the complete abolishment of the mortality from endotoxin shock (Fig. 5, A and B) and the dramatic inhibition of leukocyte (particularly eosinophil) infiltration into BAL (Fig. 5D) in the animal models examined in this study have collectively demonstrated the therapeutic efficacy of Andro for treating various inflammatory disorders. Inhibition of NF-␬B oligonucleotide binding to the nuclear proteins extracted from the lungs after LPS challenge (Fig. 5C), and the reduction of E-selectin and VCAM-1 expression in the lungs after aerosol challenge with OVA (Fig. 5F) indicate that Andro potently prevents NF-␬B activation in vivo. In addition to the long history of using Andrographis as a remedy for inflammatory disorders, previous observations that Andro suppresses NO production and down-regulates leukocyte integrin Mac-1 (␣M␤2, CD11bCD18), leading to inhibition of neutrophil adhesion and transmigration (10 –13), also strongly support this idea. Based on the structural analysis of p50, the p50 C62S mutant should have another four surface-exposed cysteines along with one pair of intramolecular disulfide-bonded cysteine residues (31–33). In this regard, the binding of Andro to wt p50 and p50 C119S mutant, but not to p50 C62S mutant (Fig. 3E), appears to suggest both the specificity of Andro/p50 interaction and the structural uniqueness of cysteine 62 in the p50 molecule. In this context, Rel homology domain reportedly constitutes three relatively flexible segments including two separable Ig-like domains. The N-terminal Ig-like domain conveys DNA binding specificity. In the N-terminal Ig-like domain, there are five base-contacting amino acids

FIGURE 6. Andro fails to inhibit I␬B␣ degradation, p50 and p65 nuclear translocation, and cell growth. A, I␬B␣ in 293 cells, without or with TNF-␣ treatment, was detected with an mAb to I␬B␣, at the indicated time points, in the absence (designated as control) or presence of Andro. B, p50 and p65 in the nuclear proteins isolated from the untreated 293 cells or the TNF-␣-treated 293 cells in the absence or the presence of the indicated amounts of Andro. C, 293 cells, NIH-3T3 cells, and HUVECs were incubated with the indicated amounts of Andro, and their growth rates were measured. Results are the mean ⫾ SD of the triplicate measurements from three independent experiments.


4216 (His64, Arg 56, Arg54, Glu60, and Lys241) in loop L1 of p50, which binds to the 5⬘ subsite (5⬘-GGGRN-3⬘) in the p50/p65 heterodimer. As cysteine 62 is also located in the loop L1 (the DNA binding pocket) of p50, it is therefore reasonable to predict that covalent conjugation of Andro to this residue will abrogate its binding activity for NF-␬B oligonucleotide. It should be pointed out that although Andro has no detectable effect on I␬B␣ degradation, p50 and p65 nuclear translocation and cell growth rates of 293 cells, NIH-3T3 cells and HUVECs (Fig. 6), our trial to directly demonstrate the binding specificity of Andro to p50 in the whole cell lysates has been confronted with technical difficulty of the minimal remaining activity of biotinylated deoxy-Andro for inhibition of NF-␬B activation after chemical conjugation of biotin (data not shown). In this regard, it should also be mentioned that Andro, but not its inactive structural analog 4H-Andro, was an equally potent inhibitor of the AP-1 transcriptional factor in both the AP-1 promoter luciferase gene reporter assay and the EMSA using AP-1 binding oligonucleotide (data not shown). These data suggest the potential pharmacological actions of Andro on another important transcriptional factor(s) or signal transduction pathway(s). However, it remains to be experimentally determined whether Andro can directly react with these transcriptional factors or signaling molecules. The oxidation reduction (redox) regulation of NF-␬B activity in vitro has been known for a decade (34). Our initial finding that DTT, a reducing reagent, could prevent the inhibitory effect of Andro on the binding of NF-␬B oligonucleotide to the isolated nuclear proteins (Fig. 2C) led us to the discovery of the covalent conjugation of Andro to the reduced cysteine 62 of p50 (Fig. 3). The preferential interaction of Andro with reduced cysteine 62 over oxidized cysteine 62 is fully consistent with the recent finding that the same amino acid, cysteine 62 of p50, is less reduced in the cytoplasm, but is strongly reduced by redox factor-1 in the nucleus, which has recently been proposed as a prerequisite for NF-␬B activation (23). Apparently, Andro mainly targets to this critical regulatory site of p50, that is, the reduced cysteine 62 of p50, for attenuation of nuclear NF-␬B transcriptional activity. Our results showing inhibition of NF-␬B activation by covalent conjugation to reduced cysteine 62 of p50 with Andro thus not only lend a support to the functional importance for the redox modulation of cysteine 62 for the interaction of p50 with NF-␬B oligonucleotide (34), but also provide a convenient tool for examining the roles of NF-␬B activation in various in vitro and in vivo models of inflammation. The subunits of p50, p65, p52, RelB, and c-Rel of the NF-␬B family form transcriptionally active dimers in a combinatorial manner, among which the p50/p65 heterodimer is most prevalent in mammalian cells. The different subunits of NF-␬B transcriptional factors apparently have distinctive roles in development and pathogenesis (1– 4, 35–37). For instance, p65⫺/⫺/p50⫺/⫺ and rel⫺/⫺/p65⫺/⫺ mice manifest embryonic death due to extensive liver apoptosis, p52⫺/⫺/p50⫺/⫺ mice manifest osteopetrosis due to a failure of osteoclast maturation and relB⫺/⫺/p50⫺/⫺ are subject to premature death within 1 mo of birth due to multiorgan inflammation. In contrast, p50⫺/⫺/rel⫺/⫺ mice are born and grow without obvious phenotypic alterations. However, the p50-null mice have a significantly reduced inflammatory response in various models of inflammation, such as asthma (37), arthritis (38), and autoimmune encephalomyelitis (39), attesting to the critical roles of p50 in inflammatory disorders. As Andro targets the reduced cysteine 62 of p50, the therapeutic potential of Andro for treating these inflammatory diseases merits further investigation in various models of inflammation. Finally, a variety of viruses, including HIV-1, HSV-1, hepatitis B virus, and hepatitis C virus can activate the NF-␬B pathway as

a mechanism of co-opting or hijacking host signaling pathways, which mediates replication of the viral genomes, induction of pathogenic responses, and suppression of apoptosis (35). For example, there are two NF-␬B sites in the HIV-1 long terminal repeat known to be directly involved in the transcription and replication of HIV-1. We speculate that Andro, given its ability to inhibit the NF-␬B pathway, may therefore prove to have anti-viral activity as well as anti-inflammatory activity.

Acknowledgments We thank Ji-Guo Liu for preparation of HUVECs, and Dr. Dolores Pe´ rez-Sala for wt p50 and p50 C62S mutant.

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