Immunomodulatory Activity of Oenothein B Isolated from Epilobium angustifolium Igor A. Schepetkin, Liliya N. Kirpotina, Larissa Jakiw, Andrei I. Khlebnikov, Christie L. Blaskovich, Mark A. Jutila This information is current as and Mark T. Quinn of September 23, 2021. J Immunol 2009; 183:6754-6766; Prepublished online 21 October 2009; doi: 10.4049/jimmunol.0901827

http://www.jimmunol.org/content/183/10/6754 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2009/10/21/jimmunol.090182 Material 7.DC1 http://www.jimmunol.org/ References This article cites 81 articles, 4 of which you can access for free at: http://www.jimmunol.org/content/183/10/6754.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

by guest on September 23, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immunomodulatory Activity of Oenothein B Isolated from Epilobium angustifolium1

Igor A. Schepetkin,* Liliya N. Kirpotina,* Larissa Jakiw,* Andrei I. Khlebnikov,† Christie L. Blaskovich,* Mark A. Jutila,* and Mark T. Quinn2*

Epilobium angustifolium has been traditionally used to treat of a number of diseases; however, not much is known regarding its effect on innate immune cells. In this study, we report that extracts of E. angustifolium activated functional responses in neutrophils and monocyte/macrophages. Activity-guided fractionation, followed by mass spectroscopy and NMR analysis, resulted in the identification of oenothein B as the primary component responsible for phagocyte activation. Oenothein B, a dimeric hydrolysable tannin, dose- ,dependently induced a number of phagocyte functions in vitro, including intracellular Ca2؉ flux, production of reactive oxygen species chemotaxis, NF-␬B activation, and proinflammatory cytokine production. Furthermore, oenothein B was active in vivo, inducing keratinocyte chemoattractant production and neutrophil recruitment to the peritoneum after intraperitoneal administration. Biological activity Downloaded from required the full oenothein B structure, as substructures of oenothein B (pyrocatechol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid) were all inactive. The ability of oenothein B to modulate phagocyte functions in vitro and in vivo suggests that this compound is responsible for at least part of the therapeutic properties of E. angustifolium extracts. The Journal of Immunology, 2009, 183: 6754–6766.

nhancement of innate immunity by immunomodulators gitannins (24). Ellagitannins are plant polyphenols, and previ-

can increase host resistance to pathogens (1), and a num- ous studies have shown that some ellagitannins (e.g., coriariin http://www.jimmunol.org/ E ber of innate immunomodulators have been identified, in- A) exhibit immunomodulatory activity (25). Oenothein B, a cluding cytokines (2), substances isolated from microorganisms dimeric macrocyclic ellagitannin, has been reported to be one of and fungi (3), and substances isolated from plants (4, 5). However, the main biologically active components in Epilobium taxa, and many of these substances are high molecular mass carbohydrates this compound is present in high concentrations in Epilobium (6) or lectins (7), and only a few plant-derived compounds with a species (24). Previous studies on oenothein B have shown that relatively low molecular mass are known to modulate phagocyte oenothein B exhibits significant antioxidant (26), antitumor (14, functions, e.g., taxol (8), phenylpropanoid glycoside acteoside (9), 27–30), antibacterial (31), and antiviral (32) activities. Al- and alkylamides from Echinacea purpurea (10). Thus, there is a though polyphenols are known for their antioxidant activity, significant amount of interest in identifying low molecular mass recent evidence indicates that the therapeutic effects of these by guest on September 23, 2021 compounds with potential medicinal properties. compounds is not solely due to antioxidant properties and that The genus Epilobium is widely distributed around the world and they can directly modulate cellular responses (reviewed in Ref. consists of over 200 species, with the most common being Epilo- 33). For example, it has been reported that oenothein B has bium angustifolium L. Various members of the genus Epilobium antitumor activity and that this may be due to enhancement of have been used in folk medicine to treat a variety of diseases and the host-immune system via induction of IL-1␤ (27). However, enhance wound healing (reviewed in Ref. 11). Indeed, extracts little else has been reported on the effects of oenothein B on from Epilobium taxa have been shown in both in vitro and in vivo innate immunity. Thus, we evaluated the effects of E. angusti- studies to exhibit many therapeutic properties, including anti- folium extracts on phagocyte function. inflammatory (12), antiandrogenic (13), antiproliferative (14, 15), In this study, we report that E. angustifolium extracts can acti- antifungal (16, 17), antimicrobial (18, 19), antinociceptive (20, vate phagocyte functional responses. Furthermore, fractionation of 21), and antioxidant (22, 23) effects. the extracts indicated that the active component was oenothein B. Although the active components responsible for therapeutic Oenothein B activated monocyte/macrophages and neutrophils, re- effects of Epilobium are not well defined, one of the classes of sulting in increased intracellular Ca2ϩ flux, production of reactive bioactive compounds present in Epilobium species is the ella- oxygen species (ROS)3 and cytokines, and chemotaxis. Thus, part of the observed therapeutic effects of oenothein B and Epilobium extracts is due to modulation of innate immune function. *Departments of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717; and †Department of Chemistry, Altai State Technical University, Barnaul, Russia Materials and Methods Received for publication June 9, 2009. Accepted for publication September 5, 2009. Reagents The costs of publication of this article were defrayed in part by the payment of page Corilagin (1-O-galloyl-3,6-hexahydroxydiphenol-␤-D-glucopyranose) was charges. This article must therefore be hereby marked advertisement in accordance from Toronto Research Chemicals; 1,2,3,4,6-pentakis-O-galloyl-␤-D-glucose with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by National Institutes of Health Grants P20 RR- 020185, P20 RR-016455, and P01 AT0004986-01; National Institutes of Health 3 Abbreviations used in this paper: ROS, reactive oxygen species; PGG, 1,2,3,4,6- contract HHSN266200400009C; an equipment grant from the M.J. Murdock Chari- pentakis-O-galloyl-␤-D-glucose; fMLF, N-formyl-Met-Leu-Phe; NBT, nitro blue tet- table Trust; and the Montana State University Agricultural Experimental Station. razolium; LAL, Limulus amebocyte lysate; KC, keratinocyte chemoattractant; FI, fold 2 Address correspondence and reprint requests to Dr. Mark T. Quinn, Veterinary increase. Molecular Biology, Montana State University, Bozeman, MT 59717. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901827 The Journal of Immunology 6755

(PGG) was from Sinova; and 8-amino-5-chloro-7-phenylpyridol [3,4- Cell culture d]pyridazine-1,4(2H,3H)-dione (L-012) was obtained from Wako Chemi- cals. Pyrocatechol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid, Human monocytic THP-1Blue cells obtained from InvivoGen were cul- Sephadex LH-20 (25–100 ␮m), DMSO, deuterium oxide (D O), EDTA, tured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 2 ␮ ␮ ␮ ionomycin, HRP, N-formyl-Met-Leu-Phe (fMLF), horseradish superoxide g/ml streptomycin, 100 U/ml penicillin, 100 g/ml Zeocin, and 10 g/ml dismutase, percoll, HEPES, Histopaque 1077, Histopaque 1119, LPS from blasticidin S. THP-1Blue cells are stably transfected with a secreted em- Escherichia coli K-235, PMA, xanthine oxidase from bovine milk, xan- bryonic alkaline phosphatase gene that is under the control of a promoter ␬ thine, nitro blue tetrazolium (NBT), and zymosan A from Saccharomyces inducible by NF- B. cerevisiae were purchased from Sigma-Aldrich. IL-8 was purchased Pepro- Human leukemia HL-60 cells were cultured in RPMI 1640 supple- mented with 10% (v/v) heat inactivated FCS, 10 mM HEPES, 100 ␮g/ml Tech. Acetone-d6 was from Cambridge Isotope Laboratories. HPLC grade acetonitrile and methanol were from EMD Chemicals (Gibbstown, NJ), streptomycin, and 100 U/ml penicillin. HL-60 cells were differentiated to macrophage-like cells by treatment with 10 nM PMA for 3 days (35). All and HPLC grade H2O and trifluoroacetic acid were from Mallinckrodt Baker. HBSS (pH 7.4), with and without Ca2ϩ and Mg2ϩ (HBSSϩ and cultured cells were grown at 37°C in a humidified atmosphere containing Ϫ HBSS , respectively), and enzyme-free cell dissociation buffer were from 5% CO2. Cell number and viability were assessed microscopically using Invitrogen. RPMI 1640 medium was purchased from Mediatech. Percoll trypan blue exclusion. stock solution was prepared by mixing Percoll with 10ϫ HBSS at a ratio Isolation of murine bone marrow leukocytes and neutrophils of 9:1. Serum-opsonized zymosan was prepared by incubating zymosan A with All animal use was conducted in accordance with a protocol approved by murine or human serum for 45 min at 25oC. After centrifugation (800 ϫ g the Institutional Animal Care and Use Committee at Montana State Uni- for 10 min), the zymosan particles were washed and resuspended in versity. Murine bone marrow cells were flushed from tibias and femurs of HBSSϪ. BALB/c mice with HBSS using a syringe with 27-gauge needle. The cells were resuspended by gentle pipetting and filtered through 70-␮m nylon cell Extraction and isolation of active compound strainers (Becton Dickinson) to remove cell clumps and bone particles, and Downloaded from resuspended in HBSSϪ at 106 cells/ml (36). This procedure minimizes Fully blossomed E. angustifolium were collected and identified by Robyn phagocyte activation during isolation and, thus, their function more accu- A. Klein (Department of Plant Sciences and Plant Pathology, Montana rately reflects the physical condition of these cells in vivo. ROS generation ϫ State University, Bozeman, MT). The dried plant material (400 g) was in bone marrow leukocyte preparations is primarily due to neutrophils and extracted with 80% methanol at room temperature for 3 days. The com- macrophages (37). bined extracts were concentrated, and any precipitates were removed by Bone marrow neutrophils were isolated from bone marrow leukocyte ␮ filtration through a 0.22- m filter. The filtrate was lyophilized to obtain the preparations, as described previously (36). Leukocyte pellets prepared as http://www.jimmunol.org/ crude extract or subjected to concentration and fractionation on a Sephadex described above were resuspended in 3 ml of 45% Percoll solution and ϫ LH-20 column (2.8 33 cm) using 80% methanol as an eluent. The rel- layered on top of a Percoll gradient consisting of 2 ml each of 50, 55, 62, evant fractions were pooled and evaporated to dryness. For analysis of and 81% Percoll solutions in a conical 15-ml polypropylene tube. The biological activity, the crude extract and pooled fractions were dissolved in gradient was centrifuged at 1600 ϫ g for 30 min at 10°C, and the cell band DMSO. Bioactive subfraction S-3 was rechromatographed twice on a located between the 61 and 81% Percoll layers was collected. The cells ϫ Sephadex LH-20 column (2.5 25 cm) using 80% methanol as the mobile were washed, layered on top of 3 ml of Histopaque 1119, and centrifuged phase, resulting in 180 mg of the final product. All lyophilized extracts and at 1600 ϫ g for 30 min at 10°C to remove contaminating RBC. The pu- Ϫ Ϫ fractions were stored at 20°C until use. For biological assays, the samples rified neutrophils were collected, washed, and resuspended in HBSS . were dissolved in HBSSϪ (ROS production and Ca2ϩ flux assays) or RPMI 1640 (cytokine production and NF-␬B activity). Isolation of human neutrophils and mononuclear cells by guest on September 23, 2021 Compound identification Blood was collected from healthy donors in accordance with a protocol approved by the Institutional Review Board at Montana State University.

For NMR analysis, samples (5 mg) were dissolved in 0.5 ml D2Oor Neutrophils and mononuclear cells were purified from the blood using 1 acetone-d6 with 2% (v/v) D2O, and H spectra were recorded on a Bruker dextran sedimentation, followed by Histopaque 1077 gradient separation DRX-600 spectrometer (Bruker) at 20°C using 3-(trimethylsilyl)-propionic and hypotonic lysis of RBC, as described previously (38). Isolated neutro- 13 Ϫ 2,2,3,3,-d4 acid sodium salt as an internal reference. For C-NMR, the phils were washed twice and resuspended in HBSS . Neutrophil prepara- spectra were recorded on a Bruker DRX-500 spectrometer at 20°C. tions were routinely Ͼ95% pure, as determined by light microscopy, and Mass spectrometry experiments were performed using a Bruker Mic- Ͼ98% viable, as determined by trypan blue exclusion. PBMC were iso- rotof high resolution time of flight mass spectrometer. Ionization was lated from blood using dextran sedimentation and Histopaque 1077 gradi- achieved using standard electrospray conditions, and data were acquired in ent separation (39). positive mode with the following settings: nitrogen drying gas temperature 200°C, drying gas flow rate 3 L/min, nebulizer pressure 0.2 bar, and cap- Analysis of phagocyte ROS production illary voltage was 4000 V. Each sample was dissolved in HPLC-grade ROS production was determined by monitoring L-012-ECL, which repre- methanol and directly infused into the mass spectrometer. The full mass Ϫ sents a sensitive and reliable method for detecting superoxide anion (O2 ) scan ranged between (m/z) 100 and 1700 Da. production in vitro (40). In brief, phagocytes were aliquoted into wells (105 Purity of isolated oenothein B was determined by reverse-phase HPLC cells/well) of 96-well flat-bottom white microplates, and test extracts or on an automated HPLC system (Shimadzu) with a Phenomenex Jupiter lyophilized fractions diluted in DMSO were added (final DMSO concen- ␮ ϫ C18 300A column (5 m, 250 4.6 mm) eluted with acetonitrile/water tration of 1%). After preincubation at 37°C for the indicated times, an equal (16/84, v/v) containing 0.1% (v/v) trifluoracetic acid at a flow rate of 1 volume of 0.05% BSA in HBSSϪ was added to each well, the plates were ml/min and detection at 270 nm, at 30°C over 15 min (34). Purity was centrifuged, the medium was removed, and fresh HBSSϩ supplemented Ͼ 95% based on reversed-phase HPLC and mass spectroscopic analysis. with 40 ␮M L-012 and 8 ␮g/ml HRP was added. In some experiments, the plates were read directly without replacing the medium to evaluate PMA- Endotoxin assays or zymosan-stimulated ROS production in the presence of the oenothein B A Limulus amebocyte lysate (LAL) assay kit (Cambrex) was used to eval- or total E. angustifolium extract. Luminescence was monitored for 60 min uate isolated oenothein B for possible endotoxin contamination, according (2-min intervals) at 37°C using a Fluroscan Ascent FL microplate reader to the manufacturer’s protocol. In brief, the LAL was reconstituted in 250 (Thermo Electron). The curve of light intensity (in relative luminescence ␮l of isolated oenothein B dissolved in endotoxin-free 0.05 M phosphate units) was plotted against time, and the area under the curve was calculated buffer (pH 7.2), and each vial was incubated at 37°C for 1 h. At the end of as total luminescence. the incubation period, each vial was inverted 180° to estimate gel forma- Xanthine/xanthine oxidase system tion compared with control (endotoxin-free buffer). Ϫ ␮ To further evaluate the possible role of endotoxin, isolated oenothein B O2 was generated in an enzymatic system consisting of 500 M xanthine, dissolved in elution buffer (0.05 M phosphate buffer (pH 7.2) containing 500 ␮M NBT, 3.75 mU/ml xanthine oxidase, and 0.1 M phosphate buffer Ϫ 0.5 M NaCl) was applied to a column containing Detoxi-Gel Endotoxin (pH 7.5), and O2 production was determined by monitoring reduction of Removing Gel (Pierce). The concentration of oenothein B in the eluted NBT to monoformazan dye at 560 nm in the presence or absence of sample was adjusted to match that of the untreated fraction, as determined oenothein B. The reactions were monitored with a SpectraMax Plus mi- by UV spectroscopy at 265 nm, and both treated and untreated samples croplate spectrophotometer at 27°C. To evaluate whether oenothein B af- Ϫ were analyzed for biological activity. fected the generation of O2 by direct interaction with xanthine oxidase, 6756 IMMUNOMODULATORY ACTIVITY OF OENOTHEIN B enzyme activity was evaluated by spectrophotometric measurement of uric jected to flow cytometric analysis using standard techniques. In brief, peri- acid formation from xanthine at 295 nm (41). The reaction mixture con- toneal cells were stained with FITC-conjugated RB6–8C5 (anti-GR-1; pan tained 500 ␮M xanthine, 5 mU/ml xanthine oxidase, and 0.1 M phosphate neutrophil stain), allophycocyanin-conjugated anti-CD45.2 (pan-leukocyte buffer (pH 7.5), and the reaction was monitored in the presence or absence stain; disregards RBC, epithelial cells, and fibrobalsts), and PE-conjugated of oenothein B at 27°C. anti-CD11b (stains macrophages and neutrophils, bright on highly acti- vated cells). All incubation and wash buffers contained 2% horse serum to 2ϩ Ca mobilization assay minimize background staining of the cell preparations. The samples were ϩ analyzed on a FACSCalibur equipped with a high-throughput sampler (BD Changes in intracellular Ca2 were measured with a FlexStation II scan- Biosciences). Viable leukocytes were gated based on forward-scatter/side- ning fluorometer using a FLIPR 3 Calcium Assay Kit (Molecular Devices). ϩ Ϫ scatter and positive staining for CD45. Of the viable CD45 leukocytes, Human and murine neutrophils or HL-60 cells, suspended in HBSS con- the percentage of neutrophils (identified by distinctive light scatter and taining 10 mM HEPES, were loaded with FLIPR Calcium 3 dye following bright positive ϩ staining of GR-1 /CD11b ) was determined. the manufacturer’s protocol. After dye loading, Ca2 was added to the cell In some experiments, serum samples were collected, and keratinocyte suspension (2.25 mM final), and cells were aliquotted into the wells of a chemoattractant (KC) levels were quantified by using a Mouse KC Quan- flat-bottom black microplate (2 ϫ 105 cells/well). The compound source tikine ELISA kit (R&D Systems). Assay plates were read at 450 nm using plate contained dilutions of E. angustifolium extract or test compounds in ϩ a Molecular Devices VERSAMax microplate reader. KC levels were de- HBSS or DMSO. Changes in fluorescence were monitored (␭ ϭ 485 ex termined by extrapolation from recombinant KC standard curves, accord- nm, ␭ ϭ 525 nm) every 5 s for 120 s at room temperature after auto- em ing to the manufacturer’s protocol. mated addition of compounds to the wells. Maximum change in fluores- cence, expressed in arbitrary units over baseline signal observed in cells ϩ Statistical analysis treated with vehicle (HBSS or DMSO), was used to determine agonist response. Curve fitting and calculation of median effective concentration Statistical analysis was performed using Prism 5. The data were analyzed values (EC ) were performed by nonlinear regression analysis of the dose- 50 by one way ANOVA, followed by Tukey’s Multiple Comparison Test, Downloaded from response curves generated using Prism 5 (GraphPad Software). with the exception of the data from in vivo experiments, which were an- alyzed by Student’s t test. Statistically significant differences ( p Ͻ 0.05 or Chemotaxis assay better) compared with the appropriate controls are indicated. Human neutrophils were suspended in HBSSϩ containing 2% (v/v) FBS (2 ϫ 106 cells/ml), and chemotaxis was analyzed in 96-well ChemoTx Results chemotaxis chambers (Neuroprobe), as described previously (42). In brief, Effect of E. angustifolium extract on ROS production and lower wells were loaded with 30 ␮l of HBSSϩ containing 2% (v/v) FBS NF-␬B activity http://www.jimmunol.org/ and E. angustifolium extract, the indicated concentrations of oenothein B Ϫ vehicle control (DMSO or HBSS ), or IL-8 and fMLF as positive controls. To evaluate the effects of E. angustifolium extracts on phagocyte The number of migrated cells was determined by measuring ATP in lysates functional responses, we analyzed the effects of methanol extracts of transmigrated cells using a luminescence-based assay (CellTiter-Glo; ␬ Promega), and luminescence measurements were converted to absolute cell from this plant on phagocyte ROS production and NF- B activa- numbers by comparison of the values with standard curves obtained with tion. As shown in Fig. 1, extracts from E. angustifolium dose- known numbers of neutrophils. The results are expressed as percentage of dependently activated ROS production in murine bone marrow negative control and were calculated as follows: (number of cells migrating leukocytes and induced NF-␬B in human THP-1 monocytes. in response to test compounds/spontaneous cell migration in response to control medium) ϫ 100. EC values were determined by nonlinear re- 50 Identification of the phagocyte-activating component in gression analysis of the dose-response curves generated using Prism 5 by guest on September 23, 2021 (GraphPad Software). E. angustifolium extract Cytokine analysis Based on these results, we fractionated the extract in efforts to identify the active component in this crude mixture. Concentrated Cells were incubated for 24 h in culture medium supplemented with 3% methanol extract from E. angustifolium was fractionated by pre- (v/v) endotoxin-free FBS, with or without compounds or LPS as a positive parative Sephadex LH-20 chromatography and sixty 10-ml frac- control. Human PBMCs and THP-1Blue human monocytic cells were plated in 96-well plates at a density of 2 ϫ 105 cells/well. A human cy- tions were collected (Fig. 2A). These fractions were pooled into tokine MultiAnalyte ELISArray Kit (SABiosciences) was used to evaluate three subfractions, designated as S-1 to S-3, and activity of these various cytokines (IL-1␣, IL-1␤, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL- subfractions was evaluated in the NF-␬B reporter assay. Only sub- ␥ ␣ 17A, IFN- , TNF- , and GM-CSF) in supernatants of PBMC or THP- fraction S-3 activated NF-␬B in this assay (Fig. 2B). 1Blue monocytes. Human TNF-␣ or IL-6 ELISA kits (BD Biosciences) were used to confirm TNF-␣ or IL-6 levels in the cell supernatants. Cy- The pooled subfraction S-3 was rechromatographed twice more tokine concentrations were determined by extrapolation from cytokine on a Sephadex LH-20 to obtain the final sample, which was con- standard curves, according to the manufacturer’s protocol. centrated to dryness and analyzed by HPLC, mass spectroscopy, and 1H- and 13C-NMR (supplemental Table S1).4 Based on com- Analysis of NF-␬B activation parison of mass spectroscopy and NMR data with those in the Activation of NF-␬B was measured using an alkaline phosphatase reporter literature (34, 43–45), we found that the active component present gene assay in THP1-Blue cells. Extracts, compounds, or LPS (100 ng/ml) ϫ 5 in subfraction S-3 isolated from E. angustifolium was oenothein B were added, the cells (2 10 cells/well) were incubated for 24 h, and ϭ alkaline phosphatase activity was measured in cell supernatants using (Mr 1568; structure of the compound is shown in supplemental QUANTI-Blue mix (InvivoGen). Activation of NF-␬B is reported as an Figure S1). absorbance at 655 nm and compared with positive control samples (LPS). As shown in supplemental Figure S2, the mass spectrum was ϩ Cytotoxicity assay characterized by the presence of monosodium (M plus Na) at m/z 1592.1 (1 m/z unit separation between isotopic peaks) and doubly Cytotoxicity was analyzed with a CellTiter-Glo Luminescent Cell Viability charged disodium (M plus 2Na)2ϩ at m/z 807.1 (0.5 m/z unit sep- Assay Kit (Promega), according to the manufacturer’s protocol. Following aration between peaks) adducts of oenothein B. The prominent treatment, the cells were allowed to equilibrate to room temperature for 30 min, substrate was added, and the samples were analyzed with a Fluo- series at m/z 1068.7 is apparently a noncovalent dimeric aggregate roscan Ascent FL. of the monosodium and disodium adducts. It should be noted that time-of-flight mass spectroscopy of polyphenols, including ellagi- In vivo analysis tannins, tends to favor association with sodium ions because nat- Oenothein B was dissolved in saline and inject i.p. into 8–12 wk old female urally occurring Naϩ ions are abundant in these samples (46, 47). BALB/c mice. At 4 h postinjection, the mice were euthanized, their peri- toneal cavities were washed with 5 ml of HBSS, and the fluid was collected and centrifuged at 500g for 5 min. The resulting cell preparation was sub- 4 The online version of this article contains supplementary material. The Journal of Immunology 6757 Downloaded from http://www.jimmunol.org/

FIGURE 1. Effect of E. angustifolium extract on ROS production in bone marrow leukocytes and NF-␬B activity in THP-1Blue monocytes. A, FIGURE 2. Fractionation of methanol extract from E. angustifolium. A, Murine bone marrow cells (105 cells/well) were treated with the indicated Crude methanol extract was isolated from florets of E. angustifolium, con- by guest on September 23, 2021 concentrations of plant extract or vehicle (1% DMSO) and incubated for 30 centrated, and fractionated using Sephadex LH-20 column chromatogra- min at 37°C. The medium was removed and replaced with HBSSϩ con- phy. Fractions were collected and pooled based on elution profile (absor- ϫ 5 taining L-012 and HRP, and chemiluminescence was monitored for 60 min. bance at 270 nm). B, THP1-Blue monocytes (2 10 cells/well) were Fold increase (FI) of integrated chemiluminescence (for 60 min) was cal- incubated for 24 h with the pooled subfractions (final concentration 50 ␮ culated in comparison with vehicle control. B, Human THP1-Blue mono- g/ml) or vehicle (1% DMSO), and alkaline phosphatase release was an- cytes (2 ϫ 105 cells/well) were incubated for 24 h with the indicated alyzed spectrophotometrically in the cell supernatant, as described. The Ϯ concentrations of plant extract or vehicle (1% DMSO). Alkaline phospha- data are presented as mean SD of triplicate samples from one experiment tase release was analyzed spectrophotometrically in the cell supernatant, as that is representative of three independent experiments. Statistically sig- Ͻ ءءء described. For both panels, the data are presented as mean Ϯ SD of trip- nificant differences ( , p 0.001) vs DMSO control are indicated. licate samples from one experiment that is representative of three indepen- p Ͻ ,ءء ;p Ͻ 0.05 ,ء) dent experiments. Statistically significant differences -p Ͻ 0.001) vs DMSO control are indicated. by the broad singlet of residual water protons, many signals char ,ءءء ;0.01 acteristic of oenothein B were observed. For example, resonances of ␣-glucose H-1 at ␦ 6.17 (d, J 3 Hz) and of ␤-glucose H-5 at ␦ 1 The H-NMR spectrum of our sample in D2O contained six 4.11 (dd, J1 5, J2 10 Hz) are very similar to the corresponding 1H-singlets and two 2H-singlets in the aromatic region (supple- signals of oenothein B reported previously [␦ 6.18 and ␦ 4.12, ␦ ␦ mental Table S1), which is in agreement with the presence of two respectively, in acetone-d6–D2O (43); 6.24 and 4.14, respec- galloyl and two valoneoyl moieties in oenothein B. Two glucopy- tively, in acetone-d6 (44)]. These values are quite distinct from any ranose residues gave well-resolved signals of sugar protons, with glucose chemical shifts in the 1H-NMR spectrum of oenothein D characteristic coupling similar to that of the oenothein B spectrum (43). Eight singlets of galloyl and valoneoyl aromatic rings in the in acetone (44). Anomeric proton doublets appeared at ␦ 4.47 (J 9 spectrum of our isolated sample were also very close (rms devia- Hz) and 5.47 (J 3.4 Hz), indicating that the anomeric hydroxyls of tion of 0.07 ppm) to the corresponding signals reported for

both glucose residues were nonacylated. Although an equilibrium oenothein B in acetone-d6 (44). Note that chemical shifts of aro- occurs between the ␣- and ␤-forms of each of the glucopyranose matic moieties differ significantly between oenothein isomers, and rings, the 1H-NMR spectrum showed that the ␣-form dominates galloyl and valoneoyl NMR signals for oenothein D and oenothein one ring, whereas the ␤-form dominates the other glucopyranose F are in quite different positions than for oenothein B (43). Taken moiety. This is in contrast to oenothein F, an isomer of oenothein together, our data demonstrate that the primary phagocyte-activat- B, where a mixture of anomeric forms for both rings results in a ing component of E. angustifolium extract is oenothein B. more complex 1H-NMR spectrum (43). Because previous NMR Effect of oenothein B on ROS production analysis of oenothein B has been performed in acedone-d6,we 1 obtained additional H-NMR spectra of our sample in acedone-d6 In studies described above, the E. angustifolium extract and sub- with 2% (v/v) D2O. Although some of the signals were overlapped fractions were removed from the cells and replaced with fresh 6758 IMMUNOMODULATORY ACTIVITY OF OENOTHEIN B

medium before analysis of ROS production by treated phagocytes. We used this approach to avoid antioxidant effects of potential plant-derived compounds, as it is known that E. angustifolium ex- tract contains flavonoids, such as myricetin, kaempferol, and quer- cetin, which have antioxidant activity (45, 48, 49). To directly evaluate this issue, we analyzed potential scavenging effects of crude E. angustifolium extract or oenothein B on ROS produced by zymosan- and PMA-stimulated murine bone marrow leukocytes and human neutrophils. We found that the ROS signal was dose- dependently decreased when crude extract (5–20 ␮g/ml; data not shown) or oenothein B were present. Indeed, the ROS signal was completely lost when oenothein B was present in the assay at con- Ͼ ␮ centrations 2–3 M (Fig. 3, A and B). IC50 values were 50 and 90 nM oenothein B for scavenging ROS produced by PMA-stim- ulated human neutrophils and murine bone marrow leukocytes, respectively, and 110 and 505 nM oenothein B for scavenging ROS produced by zymosan-stimulated human neutrophils and mu- rine bone marrow leukocytes, respectively. Because ROS are gen-

erated extracellularly by PMA-stimulated cells, whereas zymosan- Downloaded from stimulated cells generate intracellular ROS, which then can diffuse

out of the cell, it is not surprising that oenothein B had lower IC50 values for scavenging ROS in the PMA-stimulated cell system. To verify oenothein B was an effective ROS scavenger, we an- Ϫ alyzed scavenging activity in an enzymatic, O2 -generating sys-

tem. As shown in Fig. 3C, oenothein B (1–50 ␮M) effectively http://www.jimmunol.org/ Ϫ scavenged O2 in a xanthine/xanthine oxidase assay. To confirm the effect of oenothein B was due to ROS scavenging and not direct inhibition of xanthine oxidase itself, we measured xanthine oxidase-dependent production of uric acid from xanthine and found no effect of oenothein B over the entire concentration range tested (Fig. 3C). Thus, it is clear that, in addition to its phagocyte- activating properties, oenothein B is an effective scavenger of phagocyte-derived ROS, which is consistent with previous reports demonstrating oenothein B has antioxidant properties (26). by guest on September 23, 2021 To eliminate or decrease antioxidant effects of the compounds/ extract under investigation, the medium containing test samples was removed and replaced with fresh medium before subsequent analysis of ROS production. As shown in Fig. 4A, the kinetics of murine bone marrow leukocyte ROS production induced by E. angustifolium extract and isolated oenothein B were similar, and ROS production was induced in a time-dependent manner by oenothein B (Fig. 4B). Likewise, purified murine neutrophils dose- dependently generated ROS in response to oenothein B (Fig. 4C). ␮ At concentrations of 10–50 M oenothein B, the response pla- FIGURE 3. Analysis of oenothein B antioxidant activity. Human neu- teaued, which may be due to competing antioxidant activity of trophils (A) or murine bone marrow leukocytes (B) were aliquotted into compounds remaining even after medium replacement. SOD (50 microplate wells (105 cells/well) and treated with 200 nM PMA or 100 U/ml) completely (Ͼ95%) inhibited ROS production in oenothein ␮g/ml opsonized zymosan particles and the indicated concentrations of B-stimulated neutrophils (data not shown), indicating the response oenothein B. Chemiluminescence was monitored immediately for1hinthe Ϫ ␮ L was primarily due to NADPH oxidase-generated O2 . presence of 25 M -012. C, The indicated concentrations of oenothein B To evaluate the role of oenothein B structural components on were added to the xanthine/xanthine oxidase system. For analysis of anti- phagocyte activation, we evaluated ROS production by phagocytes oxidant activity, NBT reduction was determined. For analysis of xanthine treated with substructures of oenothein B, including pyrocatechol, oxidase inhibition, uric acid production was determined. For all panels, the data are presented as mean Ϯ SD of triplicate samples from one experiment gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid, and two related that is representative of three independent experiments. tannins, PGG and corilagin (structures of the compounds are shown in supplemental Figure S3). Only oenothein B and PGG activated ROS production over the concentration range tested (10– 100 ␮M). Fig. 5 shows the response to these compounds at se- E. angustifolium extract induced a dose-dependent increase in ␮ 2ϩ ␮ lected concentrations, as compared with that induced by 25 M [Ca ]i in human neutrophils (Fig. 6A), with an EC50 of 53 g/ml. oenothein B. Likewise, oenothein B induced a rapid and dose-dependent Ca2ϩ flux in human (Fig. 6A) and murine neutrophils (Fig. 6B), with Effect of E. angustifolium extract and purified oenothein B on ␮ 2ϩ EC50 values of 25.5 and 18.3 M, respectively. The peak levels phagocyte Ca mobilization and chemotaxis of intracellular Ca2ϩ were reached within 60 s of exposure to 2ϩ The ability of E. angustifolium extract and oenothein B to induce oenothein B and then decreased. Note however, that [Ca ]i were an intracellular Ca2ϩ flux in neutrophils was examined. Crude still higher than the basal levels at 3 min postexposure, which is The Journal of Immunology 6759

FIGURE 5. Effect of oenothein B and related compounds on ROS pro- 5 duction in bone marrow leukocytes. Bone marrow leukocytes (10 cells/ Downloaded from well) were preincubated with the indicated compounds or 1% DMSO (ve- hicle control) for 60 min. The medium was removed and replaced with ϩ HBSS containing L-012 and HRP, and chemiluminescence was moni- tored. Concentrations of pyrocatechol, gallic acid, pyrogallol, and 3,4-di- hydroxybenzoic acid (DHB) were 100 ␮M; concentrations of PGG and corilagin were 50 ␮M; concentration of oenothein B was 25 ␮M. PGG and

corilagin were dissolved in DMSO, whereas all other compounds were http://www.jimmunol.org/ dissolved in HBBS. The data are presented as mean Ϯ SD of triplicate samples from one experiment that is representative of three independent p Ͻ 0.001) vs ,ءءء) experiments. Statistically significant differences DMSO control are indicated.

Treatment of PMA-differentiated human HL-60 with oenothein B resulted in an intracellular Ca2ϩ flux, whereas no significant ϩ 2 by guest on September 23, 2021 changes in [Ca ]i were observed in undifferentiated HL-60 cells (Fig. 7, upper panel). In comparison, the Ca2ϩ ionophore iono- 2ϩ mycin increased [Ca ]i in both nondifferentiated and differenti- ated HL-60 cells (Fig. 7, lower panel). These findings suggest that oenothein B does not act as an ion channel and that phagocyte activation by this compound depends on phagocyte differentiation and/or level of receptor expression. Similar to the results observed for ROS production, pyrocat- echol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid, and corilagin failed to induce an intracellular Ca2ϩ flux in human and murine neutrophils, while PGG treatment resulted in in- 2ϩ ␮ creased [Ca ]i, although with a higher EC50 (31.3 M) than FIGURE 4. Effect of E. angustifolium extract and oenothein B on ROS oenothein B. production in murine bone marrow leukocytes and neutrophils. A, Murine Previous reports indicated that Ca2ϩ mobilization is associated bone marrow leukocytes (105 cells/well) were treated with extract (50 ␮g/ with chemotactic activity of various agents (50). Thus, we exam- ml) or oenothein B (25 ␮M) for 2 h. The medium was removed and re- ϩ ined the effect of E. angustifolium extract and oenothein B on placed with HBSS containing L-012 and HRP, and chemiluminescence human neutrophil chemotaxis. Both the extract (data not shown) was monitored for 60 min. Representative of three independent experi- and purified oenothein B exhibited neutrophil chemotactic activity ments. B, Bone marrow leukocytes (105 cells/well) were treated with oenothein B (12.5 ␮M). At the indicated times, the medium was removed and dose-dependently induced neutrophil migration, with EC50 ϩ ␮ ␮ and replaced with HBSS containing L-012 and HRP, and chemilumines- values of 46 g/ml and 11.8 M, respectively (Fig. 8). Note, how- cence was monitored. C, Purified murine neutrophils were treated with the ever, that the magnitude of this response was generally lower than indicated concentrations of oenothein B for 60 min, the medium was re- that induced by the positive controls, fMLF and IL-8, which had ϳ placed, and L-012-dependent chemiluminescence was recorded. For B and EC50 values of 1 and 12 nM, respectively (Fig. 8). C, the data are presented as mean Ϯ SD of triplicate samples from one experiment that is representative of three independent experiments. Effect of oenothein B on phagocyte NF-␬B activity To evaluate possible signaling pathways involved in the immuno- modulatory activity of oenothein B, we used a transcription factor- similar to the response observed in cells treated with fMLF (Fig. based bioassay for NF-␬B activation in human THP-1 monocytes. 6). If cells were treated in the absence of extracellular Ca2ϩ,no NF-␬B transcription reporter activity was time-dependently in- Ca2ϩ flux was observed, suggesting that oenothein B treatment duced by oenothein B (25 ␮M) and LPS (100 ng/ml), but with induced influx of extracellular Ca2ϩ (data not shown). slightly different kinetics for these stimuli (Fig. 9A). For example, 6760 IMMUNOMODULATORY ACTIVITY OF OENOTHEIN B Downloaded from http://www.jimmunol.org/

ϩ FIGURE 6. Representative kinetics of phagocyte Ca2ϩ mobilization af- FIGURE 7. Analysis of oenothein B-induced Ca2 mobilization in ter treatment with E. angustifolium extract and purified oenothein B. A, HL-60 cells. Upper panel, HL-60 cells were differentiated with 10 nM Human neutrophils were treated with 100 ␮g/ml E. angustifolium extract, PMA for 4 days, the cells were treated with 50 ␮M oenothein B or control ϩ 50 ␮M oenothein B, 5 nM fMLF, or HBSS and calcium flux was moni- HBSS, and Ca2 flux was monitored, as described. Lower panel, Undif- ␮ ␮ tored, as described. B, Murine neutrophils were treated with 50 M ferentiated human HL-60 cells were treated with 50 M oenothein B, 125 by guest on September 23, 2021 oenothein B, 1 ␮M fMLF, or HBSS and calcium flux was monitored, as nM ionomycin, or control HBSS and calcium flux was monitored. The data described. The data are representative of three independent experiments. are representative of three independent experiments.

statistically significant activation of the NF-␬B reporter was ob- lected concentrations, as compared with that induced by 25 ␮M served at 12 h in LPS-treated cells, whereas NF-␬B reporter ac- oenothein B. tivity induced by oenothein B only reached statistical-significance NF-␬B activation by was not due to endotoxin contamination in at 18 h. Note however, that the response does appear to be in- our samples, as analysis of oenothein B for endotoxin using a LAL creasing in oenothein B-treated cells at 12 h, and that the level of assay showed that the isolated compound contained Ͻ0.625 ng/mg NF-␬B reporter activity induced by oenothein B is equal to or even endotoxin, which is considered to be insignificant for various bio- slightly higher than that induced by LPS at 18 h. Thus, it is likely active products (51, 52). Because it is possible that oenothein B that the level of NF-␬B reporter activity reaches statistical signif- could inhibit a pathway in the LAL coagulation cascade, for ex- icance somewhere between 12 and 18 h in oenothein B-treated ample by binding to one of the protein factors, we also pretreated cells (Fig. 9A), suggesting that oenothein B induces NF-␬B with oenothein B by elution through a column of endotoxin-removing similar or slightly delayed kinetics compared with LPS. As shown gel. We found that the pretreated oenothein B (oenothein B-ER) in Fig. 9B, the response of THP-1 monocytes to oenothein B was was just as active as untreated sample (supplemental Fig. S4), dose-dependent, and the level of NF-␬B reporter activity induced again confirming the absence of endotoxin contamination. by oenothein B at concentrations of 25–50 ␮M far exceeded that induced by 100 ng/ml LPS. Importantly, these concentrations of Effect of E. angustifolium extract and purified oenothein B on oenothein B exhibited little toxicity in THP1-Blue cells over the phagocyte cytokine production 24 h assay period (Fig. 9B). Finally, treatment of cells with both Previous studies showed that oenothein B stimulated release of oenothein B and LPS together led to significantly enhanced re- IL-1␤ from murine and human macrophages (28); however, the sponses at 12, 18, and 24 h, suggesting there was an additive or effect of this compound on other cytokines has not been evaluated. possibly synergistic effect of these two agents in activating NF-␬B To address this issue, condition medium from THP-1Blue mono- and again supporting their similar kinetics of activation (Fig. 9A). cytes and human PBMCs was analyzed using a cytokine ELISA Pyrocatechol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic semiquantitative array. Among the 12 cytokines analyzed, five acid, carilagin, and PGG were also evaluated for activity in the were consistently induced in PBMCs (Ͼ10-fold) by 10 ␮M oenothein NF-␬B alkaline phosphatase reporter assay. All of these com- B, as compared with control cells. These included IFN-␥ (fold in- pounds, including PGG, failed to induce NF-␬B reporter activity crease (FI) ϭ 11), IL-1␤ (FI ϭ 13), GM-CSF (FI ϭ 15), TNF-␣ (FI ϭ when tested in the concentration range of 10–100 ␮M. Supple- 31), and IL-6 (FI ϭ 34) (Fig. 10A). IL-8 production was inconclu- mental Figure S4 shows the response to these compounds at se- sive because of high background production by PBMCs (data The Journal of Immunology 6761

FIGURE 8. Human neutrophil chemotactic response to oenothein B. Neutrophil chemotaxis in response to the indicated concentrations of oenothein B, fMLF, and IL-8 was analyzed in chemotaxis chambers, as described. The data are presented as the number of migrated cells (mean Ϯ SD; n ϭ 3). A representative experiment from three independent experiments is shown. not shown), a problem which has also been documented previously To quantify dose-dependent effects of E. angustifolium extract (e.g., Ref. 53). Human monocytic THP-1Blue cells, treated with 25 and oenothein B on cytokine production, levels of TNF-␣ and IL-6 Downloaded from ␮M oenothein B produced high levels of TNF-␣ (FI ϭ 24), IL-6 were determined by ELISA of supernatants from treated cells. Un- (FI ϭ 94), and IL-8 (FI ϭ 98) (Fig. 10A). treated cells produced negligible amounts of TNF-␣ and IL-6, whereas incubation of THP-1Blue monocytes and PBMCs with crude extract (Fig. 10, B and D) or purified oenothein B (Fig. 10, C and E) enhanced TNF-␣ and IL-6 production in a dose-depen-

dent manner. Note, however, that pretreatment of oenothein B with http://www.jimmunol.org/ endotoxin-removing gel no effect on its activity, indicating that the induction of TNF-␣ and IL-6 production was not due to endotoxin contamination (data not shown).

In vivo analysis of oenothein B To evaluate the effect of oenothein B in vivo, mice were treated by intraperitoneal injection of oenothein B, and neutrophil recruit-

ment to the peritoneum was evaluated. As shown in Fig. 11, by guest on September 23, 2021 oenothein B significantly induced neutrophil recruitment (ϳ10- fold increase over saline controls). Because the mice were ana- lyzed 4-h postinjection, neutrophils were the primary phagocyte recruited. As expected, little change in monocyte/macrophage lev- els was observed in this short period of time (data not shown). The primary neutrophil chemotactic agent at inflammatory sites is IL-8 (KC in mice). Thus, we evaluated KC levels in mice treated with oenothein B and found that oenothein B dose-dependently induced significant levels of serum KC, which correlated with the observed recruitment of neutrophils into the peritoneum (Fig. 12). Thus, these studies verify our in vitro experiments and confirm that this oenothein B is active in vivo.

Discussion Extracts from E. angustifolium have been reported to be beneficial for treating a variety of medical problems, such as gastrointestinal, and prostate diseases, and to improve the healing of wounds (11). FIGURE 9. Effect of oenothein B on NF-␬B activity and cell viability in However, little is known regarding the effects of E. angustifolium THP1-Blue monocytes. A, THP1-Blue monocytes (2 ϫ 105 cells/well) on innate immune functions. In this study, we demonstrate that E. were incubated for the indicated times with control medium, 25 ␮M angustifolium extracts can induce or enhance phagocyte functional oenothein B, 100 ng/ml LPS, or 25 ␮M oenothein B plus 100 ng/ml LPS. responses and that the active principle in these extracts is Alkaline phosphatase release was analyzed spectrophotometrically in the oenothein B. Because oenothein B is a major component of Epi- p Ͻ lobium (24), we propose that the effects of oenothein B on innate ,ءء ;p Ͻ 0.05 ,ء) cell supernatant. Statistically significant differences Ͻ ءءء 0.01; , p 0.001) vs medium control are indicated. B, THP1-Blue immune function likely contribute to the therapeutic efficacy of monocytes (2 ϫ 105 cells/well) were incubated for 24 h with the indicated Epilobium extracts. concentrations of oenothein B or LPS, and alkaline phosphatase release was analyzed spectrophotometrically in the cell supernatant. Cell viability Oenothein B was first isolated from Oenothera erythrosepala was also determined using a CellTiter-Glo Luminescent Cell Viability As- (Onagraceae) (54) and was subsequently found in Eucalyptus, Eu- say Kit. For both panels, the data are presented as mean Ϯ SD of triplicate genia species, and Lythraceae species (31). Previous studies on samples from one experiment that is representative of three independent oenothein B have shown that it exhibits significant antioxidant experiments. (26), antitumor (27, 28, 30, 55), antibacterial (56), and antiviral 6762 IMMUNOMODULATORY ACTIVITY OF OENOTHEIN B

FIGURE 10. Effect of E. angusti- folium extract and oenothein B on cy- tokine production by human THP- 1Blue monocytes and PBMCs. A, THP-1Blue cells and PBMCs were incubated for 24 h with 25 ␮M oenothein B, and production of cyto- kines in the supernatants was evalu- ated using a MultiAnalyte ELISArray Downloaded from kit. Cytokine expression is shown as an OD (A450-A570) ratio normalized to background. B–E, THP-1Blue cells and PBMCs were incubated for 24 h with the indicated concentrations E. angustifolium extract (B and D)or oenothein B (C and E). Cell-free su- http://www.jimmunol.org/ pernatants were collected, and se- creted TNF-␣ (B and C) and IL-6 (D and E) were quantified by ELISA. For B–E, the data are presented as mean Ϯ SD of triplicate samples from one experiment that is representative of three independent experiments. by guest on September 23, 2021

(32) activities, although the mechanisms involved are not well de- of the observed therapeutic effects of oenothein B and Epilobium fined. Most studies on oenothein B have focused on its antitumor extracts in general are due to enhancement of innate immune activity, and it has been shown to inhibit poly-(ADP-ribose) gly- responses. cohydrolase, 5␣-reductase, and aromatase (24, 57, 58), and also to In addition to enhancement of innate immune function, induce a neutral endopeptidase in prostate cancer cells (34). Miy- oenothein B also was found to scavenge ROS generated by acti- amoto et al. (27) reported that oenothein B had potent antitumor vated phagocytes or by an enzymatic system, which confirms pre- activity upon intraperitoneal administration to mice before tumor vious reports on the antioxidant activity of this compound deter- inoculation and suggested that this may be due to enhancement of mined using a radical scavenging assay (26, 45). Thus, oenothein the host-immune system via macrophage activation. However, es- B is able to stimulate local innate immunity but may also protect sentially nothing else has been reported on the effects of oenothein tissues from excessive ROS production. Although the antioxidant B on innate immunity. We show in this study that oenothein B activity of polyphenols has been assumed to be the primary ther- activates phagocytic cells, including monocyte/macrophages and apeutic property, recent studies indicate that many polyphenols neutrophils, resulting in increased intracellular Ca2ϩ, production directly impact cellular signaling events, which is in independent of ROS and cytokines, and chemotactic activity. Additionally, we of their antioxidant activity (e.g., see Ref. 33). Because antioxidant demonstrated that oenothein B induced IL-8 production and neu- capacity is often diminished or even lost during absorption in vivo, trophil recruitment to the peritoneum of treated mice. Given the the primary therapeutic properties of oenothein B and other poly- critical role played by phagocytes in innate immunity against phenols may indeed be due to direct modulation of cellular activ- pathogens and their contribution to tumor cell destruction (re- ity, such as the modulation of innate immune functions shown in viewed in Ref. 59), our data support the possibility that at least part this study. The Journal of Immunology 6763 Downloaded from http://www.jimmunol.org/ FIGURE 11. Neutrophil recruitment to the peritoneum in response to oenothein B. BALB/c mice were injected i.p. with oenothein B (100 ␮g) or control saline, as indicated. After 4 h, peritoneal cells were collected and stained for flow cytometric analysis. Upper panel, Representative exam- FIGURE 12. Oenothein B-induced recruitment of neutrophils in vivo is ples of two-color dot plots comparing CD11b and GR-1 staining on CD45ϩ associated with induction of KC production. BALB/c mice were injected cells from saline- and oenothein B-treated mice. The quadrant containing i.p. with either 100 or 400 ␮g of oenothein B or control saline, as indicated. the neutrophil population is indicated (arrow), which was used to determine After 4 h, serum was collected for KC analysis by ELISA (A), and peri- the percentage of the overall CD45ϩ cell population in the peritoneum. toneal cells were collected and stained for flow cytometric analysis of ϩ Lower panel, Pooled results from three mice in each test group, as analyzed percent neutrophils (RB6-8C5 bright, CD45 cells) in the peritoneal Ϯ above. The data are presented as mean Ϯ SEM of three mice. Represen- washes, as described under Fig. 12 (B). The data are presented as mean by guest on September 23, 2021 tative of two independent experiments. Statistically significant difference SD of four mice. Representative of two independent experiments. Statis- .p Ͻ 0.01) vs saline control are indicated ,ءء) p Ͻ 0.01) vs saline control is indicated. tically significant differences ,ءء)

The nonspecific nature of immunomodulators makes them at- role in receptor-mediated intracellular signaling events (50). Fur- tractive because they can be used to treat a broad-spectrum of thermore, Ca2ϩ mobilization is required for ROS production in infections and are not susceptible to antibiotic resistance (60). In phagocytes, mainly through the activation of NADPH oxidase general, immunomodulators mimic the natural mechanisms used (e.g., Ref. 63). We found that oenothein B induced a transient 2ϩ by pathogens to stimulate innate immunity and thus are potentially elevation of [Ca ]i in neutrophils, indicating this compound is a beneficial in preventing infection (60). Thus, the balance between phagocyte agonist; however, compounds structurally related to the therapeutic and proinflammatory properties is important to con- building blocks of oenothein B (pyrocatechol, gallic acid, pyro- sider when evaluating immunomodulators, and the goal is to en- gallol, and 3,4-dihydroxybenzoic acid) had did not induce Ca2ϩ hance or prime local host defense without inducing excessive or mobilization or other phagocyte responses. In contrast, one of the systemic inflammation. This balance is dependent on pharmaco- two tannins tested (PGG) did induce a Ca2ϩ and ROS production dynamic and pharmacokinetic properties of the compound and in neutrophils. Although various tannins and other compounds must be determined empirically. For example, CpG DNA, an im- with galloyl groups, including galloylpunicalagin, woodfordin, munomodulator with therapeutic promise, induces a range of and cotton-derived tannins, have been reported previously to ac- phagocyte inflammatory responses via TLR9, which leads to tivate Ca2ϩ flux in phagocytes (64–67), this is the first report beneficial Th1-type responses (60, 61). Conversely, excessive evaluating the effects of oenothein B on phagocyte Ca2ϩ mobili- activation of TLR9 can contribute to detrimental inflammatory zation, ROS production, and chemotaxis. states (e.g., Ref. 62). Likewise, we suggest that therapeutic con- A number of ellagitannins and other tannins have been shown to centrations of oenothein B could prime or enhance innate im- activate phagocyte functions, including cytokine production and mune cells without inducing adverse inflammatory responses, phagocytosis. For example, the Ellagitannin geraniinm, which is which is supported by our in vivo experiments. In contrast, our isolated from Geranium funbergii, has been reported to induce in vitro data suggest that treatment with high concentrations of macrophage phagocytosis (68). Likewise, cuphiin D1, tellimagran- oenothein B could lead to excessive inflammation if sufficient dins I and II, rugosin A, casuarictin, coriariin, agrimoniin, and local concentrations were achieved. Thus, further analyses are PGG have been shown to induce IL-1␤ and/or TNF-␣ production clearly needed to further evaluate the specific pharmacological by human peripheral mononuclear cells and macrophages in vitro properties of oenothein B in vivo. (28, 69–71). We also found that oenothein B induced IL-1␤ and Initiation of intracellular Ca2ϩ flux is one of the earliest events TNF-␣ production by human monocyte/macrophages, but also associated with phagocyte receptor activation and plays a central provide the novel finding that IFN-␥, GM-CSF, IL-6, and IL-8 are 6764 IMMUNOMODULATORY ACTIVITY OF OENOTHEIN B

also induced in human monocyte/macrophages. Additionally, we As reported previously, oenothein B is a major component of found that intraperitoneal administration of oenothein B induced a Epilobium extracts and can be present at concentrations up to 14% significant level of KC in treated mice and that KC induction di- (ϳ9 ␮M in 100 ␮g/ml extract), although the level varies between rectly correlated with neutrophil influx into the peritoneum, dem- species and is reported to be 4.5% in E. angustifolium (24). How- onstrating that cytokine induction defined in this report is relevant ever, it has not yet been determined whether these concentrations in vivo. Thus, the ability of oenothein B to induce these cytokines vary in different regions of the world or between different lots of may also play an important role in the microbicidal, viricidal, and plants. We found that E. angustifolium extracts induced intracel- ϩ antitumor effects of this compound. For example, IL-1␤ is capable lular Ca2 flux, ROS production, chemotaxis, and cytokine pro- of upregulating the activity of tumoricidal NK cells and inducing duction in qualitatively similar patterns as purified oenothein B, antitumor reactivity in the regional lymph nodes and spleen (re- supporting the conclusion that oenothein B is indeed the active viewed in Ref. 72). Among the proinflammatory cytokines, IL-6 is component in these extracts. However, it is also apparent that the one of the most important mediators of fever and the acute-phase extracts were more potent than oenothein B if we use the estimated response (73). TNF-␣ has direct in vitro and in vivo cytostatic and concentration of 4.5% in our extracts (ϳ3 ␮M oenothein B in 100 cytocidal effects and, together with IL-6, is also considered as a ␮g/ml extract). The reasons for this difference are not clear; how- major immune and inflammatory mediator (73). One of the most ever, this observation is supported by studies from Kiss et al. (34) ϳ prominent characteristics of TNF-␣ is its ability to cause apoptosis who showed that E. angustifolium extract was 10-fold more of tumor cells, resulting in tumor necrosis (74). TNF-␣ also plays potent that pure oenothein B in inducing neutral endopeptidase a pivotal role in host defense and can act on macrophages in an activity in prostate cancer cells. One possibility is that other autocrine manner to enhance various functional responses and in- constituents in the extract may help to increase oenothein B Downloaded from duce the expression of a number of other immunoregulatory and bioavailability or stability. For example, the solubility of the inflammatory mediators (75). active component in a crude extract can often be reduced when NF-␬B is activated in response to stimulation by inflammatory the component is purified. It is also possible that some reactive agents, including LPS, and activation of NF-␬B is an essential step groups are altered during purification, thus affecting activity. in inducing proinflammatory cytokines, chemokines, inflammatory These issues are currently under investigation. enzymes, adhesion molecules, receptors, and inhibitors of apopto- Overall, our studies demonstrate that oenothein B activates a http://www.jimmunol.org/ number of phagocyte functions, including Ca2ϩ, NADPH oxidase sis (reviewed in Ref. 76). Treatment of phagocytes with oenothein activity, chemotaxis, and cytokine production. In addition, we es- B resulted in the activation of NF-␬B. In addition, treatment of tablished that oenothein B can modulate phagocyte activity in cells with both oenothein B and LPS resulted in an even greater vivo. The ability of this compound to modulate innate immune NF-␬B response, suggesting a synergistic effect. Therefore, the functions suggests that at least part of the reported effects of Epi- ability to activate phagocyte NF-␬B signaling provides further ev- lobium extracts and purified oenothein B on wound healing and idence that oenothein B possesses immunomodulatory properties. inhibition of tumor growth is through modulation of macrophage The synergistic effect of oenothein B and LPS is consistent with function. These studies suggest that oenothein B may serve as a

studies of Feldman (25), who suggested that dimeric tannins could by guest on September 23, 2021 promising lead for further therapeutic development. mimic the lipid A moiety of LPS. In contrast with our data, Chen et al. (77) reported that oenothein B modestly inhibited LPS-in- duced NF-␬B activity in Bcl-2-overexpressing murine RAW264.7 Acknowledgments macrophages. Although the reasons for this difference are not We thank Dr. Robyn Klein (Department of Plant Sciences and Plant Pa- thology, Montana State University, Bozeman, MT) for plant identification. clear, it is possible that this may be due to differences in the cell ␬ We would also thank Drs. Scott Busse and Philip Clark (Department of lines used. Alternatively, NF- B activation by oenothein B could Chemistry, Montana State University, Bozeman, MT) for help in running be indirect and mediated by cytokines induced during the assay, as NMR and mass spectroscopy samples. NF-␬B reporter activity only began to increase at after 12 h incu- bation with oenothein B and was not significant until 18 h. Thus, Disclosures further studies are now in progress to evaluate this issue and de- The authors have no financial conflict of interest. termine which receptor(s) and intercellular pathway(s) are in ␬ NF- B activation and expression of various cytokines induced by References oenothein B. 1. Ballow, M., and R. Nelson. 1997. Immunopharmacology: immunomodulation Oenothein B and a related tannin, PGG, induced ROS produc- and immunotherapy. J. Am. Med. Assoc. 278: 2008–2017. tion and Ca2ϩ mobilization in phagocytes, whereas, the mono- 2. Villinger, F. 2003. Cytokines as clinical adjuvants: how far are we? Expert. Rev. Vaccines 2: 317–326. meric polyphenols pyrocatechol, gallic acid, pyrogallol, and 3,4- 3. Wasser, S. P. 2002. Medicinal mushrooms as a source of antitumor and immu- dihydroxybenzoic acid were inactive. On the other hand, only nomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60: 258–274. oenothein B, which has a dimeric macrocyclic structure (see sup- 4. Paulsen, B. S. 2001. Plant polysaccharides with immunostimulatory activities. Curr. Organic Chem. 5: 939–950. plemental Fig. S1), induced NF-␬B activity. In addition, HL-60 5. Kayser, O., K. N. Masihi, and A. F. Kiderlen. 2003. Natural products and syn- cells responded to oenothein B only after differentiation. These thetic compounds as immunomodulators. Expert. Rev. Anti. Infect. Ther. 1: 319–335. findings suggest oenothein B may be activating phagocytes 6. Schepetkin, I. A., and M. T. Quinn. 2006. Botanical polysaccharides: macrophage through a specific receptor or cellular target, which is yet to be immunomodulation and therapeutic potential. Int. Immunopharmacol. 6: identified. Tannins bind to a wide range of targets, including 317–333. 7. Gabius, H. J. 2001. Probing the cons and pros of lectin-induced immunomodu- phospholipids, carbohydrates, and proteins (78–80). For exam- lation: case studies for the mistletoe lectin and galectin-1. Biochimie 83: ple, Teng et al. (81) reported that the ellagitannin rugosin E acti- 659–666. vated platelet Ca2ϩ flux by acting as an ADP receptor agonist. 8. Byrd-Leifer, C. A., E. F. Block, K. Takeda, S. Akira, and A. Ding. 2001. The role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol. Eur. J. Immunol. 31: Thus, it is possible that oenothein B could be interacting with a 2448–2457. number of extracellular membrane targets, such as receptors or 9. Fujinaga, Y., K. Inoue, T. Nomura, J. Sasaki, J. C. Marvaud, M. R. Popoff, S. Kozaki, and K. Oguma. 2000. Identification and characterization of functional lipid rafts involved in regulating phagocyte activation. Neverthe- subunits of Clostridium botulinum type A progenitor toxin involved in binding to less, further work is necessary to identify the target of oenothein B. intestinal microvilli and erythrocytes. FEBS Lett. 467: 179–183. The Journal of Immunology 6765

10. Gertsch, J., R. Schoop, U. Kuenzle, and A. Suter. 2004. Echinacea alkylamides that induce macrophage tumor necrosis factor ␣ production. Mol. Pharm. 74: modulate TNF-␣ gene expression via cannabinoid receptor CB2 and multiple 392–402. signal transduction pathways. FEBS Lett. 577: 563–569. 40. Daiber, A., M. August, S. Baldus, M. Wendt, M. Oelze, K. Sydow, 11. Tyler, V. E. 1986. Some recent advances in herbal medicines. Pharmacy Int. 7: A. L. Kleschyov, and T. Munzel. 2004. Measurement of NAD(P)H oxidase- 203–207. derived superoxide with the luminol analogue L-012. Free Radical Biol. Med. 36: 12. Hiermann, A., H. Juan, and W. Sametz. 1986. Influence of Epilobium extracts on 101–111. prostaglandin biosynthesis and carrageenin induced oedema of the rat paw. 41. Bindoli, A., M. Valente, and L. Cavallini. 1985. Inhibitory action of quercetin on J. Ethnopharmacol. 17: 161–169. xanthine oxidase and xanthine dehydrogenase activity. Pharmacol. Res. Com- 13. Hiermann, A., and F. Bucar. 1997. Studies of Epilobium angustifolium extracts mun. 17: 831–839. on growth of accessory sexual organs in rats. J. Ethnopharmacol. 55: 179–183. 42. Schepetkin, I. A., L. N. Kirpotina, A. I. Khlebnikov, and M. T. Quinn. 2007. 14. Vitalone, A., M. Guizzetti, L. G. Costa, and B. Tita. 2003. Extracts of various High-throughput screening for small-molecule activators of neutrophils: identifica- species of Epilobium inhibit proliferation of human prostate cells. J. Pharm. tion of novel N-formyl peptide receptor agonists. Mol. Pharm. 71: 1061–1074. Pharmacol. 55: 683–690. 43. Yoshida, T., T. Chou, T. Shingu, and T. Okuda. 1995. Oenothein-D, oenothein-F 15. Vitalone, A., J. McColl, D. Thome, L. G. Costa, and B. Tita. 2003. Character- and oenothein-G, hydrolyzable tannin dimers from Oenothera laciniata. Phyto- ization of the effect of Epilobium extracts on human cell proliferation. Pharma- chemistry 40: 555–561. cology 69: 79–87. 44. Santos, S. C., and P. G. Waterman. 2001. Polyphenols from Eucalyptus consid- 16. Jones, N. P., J. T. Arnason, M. Abou-Zaid, K. Akpagana, P. Sanchez-Vindas, and eniana and Eucalyptus viminalis. Fitoterapia 72: 95–97. M. L. Smith. 2000. Antifungal activity of extracts from medicinal plants used by 45. Hevesi To´th, B., B. Blazics, and A. Ke´ry. 2009. Polyphenol composition and First Nations Peoples of Eastern Canada. J. Ethnopharmacol. 73: 191–198. antioxidant capacity of Epilobium species. J. Pharm. Biomed. Anal. 49: 26–31. 17. Webster, D., P. Taschereau, R. J. Belland, C. Sand, and R. P. Rennie. 2008. 46. Reed, J. D., C. G. Krueger, and M. M. Vestling. 2005. MALDI-TOF mass spec- Antifungal activity of medicinal plant extracts; preliminary screening studies. trometry of oligomeric food polyphenols. Phytochemistry 66: 2248–2263. J. Ethnopharmacol. 115: 140–146. 47. Hager, T. J., L. R. Howard, R. Liyanage, J. O. Lay, and R. L. Prior. 2008. 18. Battinelli, L., B. Tita, M. G. Evandri, and G. Mazzanti. 2001. Antimicrobial Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and activity of Epilobium spp. extracts. Farmaco 56: 345–348. MALDI-TOF-MS. J. Agric. Food Chem. 56: 661–669. 48. Hiermann, A., M. Reidlinger, H. Juan, and W. Sametz. 1991. Isolation of the 19. Rauha, J. P., S. Remes, M. Heinonen, A. Hopia, M. Kahkonen, T. Kujala, Downloaded from K. Pihlaja, H. Vuorela, and P. Vuorela. 2000. Antimicrobial effects of Finnish antiphlogistic principle from Epilobium angustifolium. Planta Med. 57: 357–360. plant extracts containing flavonoids and other phenolic compounds. Int. 49. Bazylko, A., A. K. Kiss, and J. Kowalski. 2007. High-performance thin-layer J. Food Microbiol. 56: 3–12. chromatography method for quantitative determination of oenothein B and quer- 20. Pourmorad, F., M. A. Ebrahimzadeh, M. Mahmoudi, and S. Yasini. 2007. An- cetin glucuronide in aqueous extract of Epilobii angustifolii herba. J. Chro- tinociceptive activity of methanolic extract of Epilobium hirsutum. Pak. J. Biol. matogr. A. 1173: 146–150. Sci. 10: 2764–2767. 50. Lew, P. D. 1990. Receptors and intracellular signaling in human neutrophils. Am. 21. Tita, B., H. Abdel-Haq, A. Vitalone, G. Mazzanti, and L. Saso. 2001. Analgesic Rev. Respir. Dis. 141: S127–S131. properties of Epilobium angustifolium, evaluated by the hot plate test and the 51. Jahr, T. G., L. Ryan, A. Sundan, H. S. Lichenstein, G. Skjak-Braek, and writhing test. Farmaco 56: 341–343. T. Espevik. 1997. Induction of tumor necrosis factor production from monocytes http://www.jimmunol.org/ 22. Shikov, A. N., E. A. Poltanov, H. J. Dorman, V. G. Makarov, V. P. Tikhonov, and stimulated with mannuronic acid polymers and involvement of lipopolysaccha- R. Hiltunen. 2006. Chemical composition and in vitro antioxidant evaluation of ride-binding protein, CD14, and bactericidal/permeability-increasing factor. In- commercial water-soluble willow herb (Epilobium angustifolium L.) extracts. fect. Immun. 65: 89–94. J. Agric. Food Chem. 54: 3617–3624. 52. Catchpole, B., A. S. Hamblin, and N. A. Staines. 2002. Autologous mixed lym- Autoim- 23. Stajner, D., B. M. Popovic, and P. Boza. 2007. Evaluation of willow herb’s phocyte responses in experimentally-induced arthritis of the Lewis rat. munity (Epilobium angustofolium L.) antioxidant and radical scavenging capacities. Phy- 35: 111–117. tother. Res. 21: 1242–1245. 53. Kikkert, R., E. R. de Groot, and L. A. Aarden. 2008. Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants. 24. Ducrey, B., A. Marston, S. Gohring, R. W. Hartmann, and K. Hostettmann. 1997. J. Immunol. Methods 336: 45–55. Inhibition of 5 ␣-reductase and aromatase by the ellagitannins oenothein A and oenothein B from Epilobium species. Planta Med. 63: 111–114. 54. Hatano, T., T. Yasuhara, M. Matsuda, K. Yazaki, T. Yoshida, and T. Okuda. 1990. Oenothein B, a dimeric, hydrolyzable tannin with macrocyclic structure,

25. Feldman, K. S. 2005. Recent progress in ellagitannin chemistry. Phytochemistry by guest on September 23, 2021 and accompanying tannins from Oenothera erythrosepala. J. Chem. Soc. Perkin 66: 1984–2000. Trans. 1: 2735–2743. 26. Yoshimura, M., Y. Amakura, M. Tokuhara, and T. Yoshida. 2008. Polyphenolic 55. Wang, C. C., L. G. Chen, and L. L. Yang. 1999. Antitumor activity of four compounds isolated from the leaves of Myrtus communis. Nat. Med. 62: macrocyclic ellagitannins from Cuphea hyssopifolia. Cancer Lett. 140: 195–200. 366–368. 56. Yoshida, T., T. Hatano, and H. Ito. 2000. Chemistry and function of vegetable 27. Miyamoto, K., M. Nomura, M. Sasakura, E. Matsui, R. Koshiura, T. Murayama, polyphenols with high molecular weights. Biofactors 13: 121–125. T. Furukawa, T. Hatano, T. Yoshida, and T. Okuda. 1993. Antitumor activity of 57. Aoki, K., H. Maruta, F. Uchiumi, T. Hatano, T. Yoshida, and S. Tanuma. 1995. oenothein B, a unique macrocyclic ellagitannin. Jpn. J. Cancer Res. 84: 99–103. A macrocircular ellagitannin, oenothein B, suppresses mouse mammary tumor 28. Miyamoto, K., T. Murayama, M. Nomura, T. Hatano, T. Yoshida, T. Furukawa, gene expression via inhibition of poly(ADP-ribose) glycohydrolase. Biochem. R. Koshiura, and T. Okuda. 1993. Antitumor activity and interleukin-1 induction Biophys. Res. Commun. 210: 329–337. Anticancer Res. by tannins. 13: 37–42. 58. Lesuisse, D., J. Berjonneau, C. Ciot, P. Devaux, B. Doucet, J. F. Gourvest, 29. Yang, L. L., C. C. Wang, K. Y. Yen, T. Yoshida, T. Hatano, and T. Okuda. 1999. B. Khemis, C. Lang, R. Legrand, M. Lowinski, et al. 1996. Determination of Antitumor activities of ellagitannins on tumor cell lines. Basic Life Sci. 66: oenothein B as the active 5-␣-reductase-inhibiting principle of the folk medicine 615–628. Epilobium parviflorum. J. Nat. Prod. 59: 490–492. 30. Sakagami, H., Y. Jiang, K. Kusama, T. Atsumi, T. Ueha, M. Toguchi, I. Iwakura, 59. Hume, D. A. 2006. The mononuclear phagocyte system. Curr. Opin. Immunol. K. Satoh, H. Ito, T. Hatano, and T. Yoshida. 2000. Cytotoxic activity of hydro- 18: 49–53. lyzable tannins against human oral tumor cell lines: a possible mechanism. Phy- 60. Finlay, B. B., and R. E. W. Hancock. 2004. Can innate immunity be enhanced to tomedicine 7: 39–47. treat microbial infections? Nat. Rev. Microbiol. 2: 497–504. 31. Yoshida, T., T. Hatano, and H. Ito. 2000. Chemistry and function of vegetable 61. Barchet, W., V. Wimmenauer, M. Schlee, and G. Hartmann. 2008. Accessing the polyphenols with high molecular weights. Biofactors 13: 121–125. therapeutic potential of immunostimulatory nucleic acids. Curr. Opin. Immunol. 32. Fukuchi, K., H. Sakagami, T. Okuda, T. Hatano, S. Tanuma, K. Kitajima, 20: 389–395. Y. Inoue, S. Inoue, S. Ichikawa, M. Nonoyama, et al. 1989. Inhibition of herpes 62. Franklin, B. S., P. Parroche, M. A. Ataide, F. Lauw, C. Ropert, R. B. de Oliveira, simplex virus infection by tannins and related compounds. Antiviral Res. 11: D. Pereira, M. S. Tada, P. Nogueira, L. H. da Silva, et al. 2009. Malaria primes 285–297. the innate immune response due to interferon-␥ induced enhancement of toll-like 33. Virgili, F., and M. Marino. 2008. Regulation of cellular signals from nutritional receptor expression and function. Proc. Natl. Acad. Sci. USA 106: 5789–5794. molecules: a specific role for phytochemicals, beyond antioxidant activity. Free 63. Sakaguchi, N., M. Inoue, and Y. Ogihara. 1998. Reactive oxygen species and Radical Biol. Med. 45: 1205–1216. intracellular Ca2ϩ, common signals for apoptosis induced by gallic acid. Bio- 34. Kiss, A., J. Kowalski, and M. F. Melzig. 2006. Induction of neutral endopeptidase chem. Pharmacol. 55: 1973–1981. activity in PC-3 cells by an aqueous extract of Epilobium angustifolium 64. Lauque, D., M. C. Prevost, P. Carles, and H. Chap. 1991. Signal transducing L. and oenothein B. Phytomedicine 13: 284–289. mechanisms in human platelets stimulated by cotton bract tannin. Am. J. Respir. 35. Jasek, E., J. Mirecka, and J. A. Litwin. 2008. Effect of differentiating agents Cell Mol. Biol. 4: 65–71. (all-trans retinoic acid and phorbol 12-myristate 13-acetate) on drug sensitivity of 65. Bates, P. J., N. V. Ralston, Z. Vuk-Pavlovic, and M. S. Rohrbach. 1995. Calcium HL60 and NB4 cells in vitro. Folia Histochem. Cytobiol. 46: 323–330. influx is required for tannin-mediated arachidonic acid release from alveolar mac- 36. Siemsen, D. W., I. A. Schepetkin, L. N. Kirpotina, B. Lei, and M. T. Quinn. 2007. rophages. Am. J. Physiol. 268: L33–L40. Neutrophil isolation from nonhuman species. Methods Mol. Biol. 412: 21–34. 66. Liu, M. J., Z. Wang, H. X. Li, R. C. Wu, Y. Z. Liu, and Q. Y. Wu. 2004. 37. Li, W., and S. C. Chung. 2003. Flow cytometric evaluation of leukocyte function Mitochondrial dysfunction as an early event in the process of apoptosis induced in rat whole blood. In Vitro Cell Dev. Biol. Anim. 39: 413–419. by woodfordin I in human leukemia K562 cells. Toxicol. Appl. Pharmacol. 194: 38. Schepetkin, I. A., A. I. Khlebnikov, and M. T. Quinn. 2007. N-Benzoylpyrazoles 141–155. are novel small-molecule inhibitors of human neutrophil elastase. J. Med. Chem. 67. Chen, L. G., Y. C. Liu, C. W. Hsieh, B. C. Liao, and B. S. Wung. 2008. Tannin 50: 4928–4938. 1-␣-O-galloylpunicalagin induces the calcium-dependent activation of endothe- 39. Schepetkin, I. A., L. N. Kirpotina, J. Tian, A. I. Khlebnikov, R. D. Ye, and lial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in M. T. Quinn. 2008. Identification of novel formyl peptide receptor-like 1 agonists endothelial cells. Mol. Nutr. Food Res. 52: 1162–1171. 6766 IMMUNOMODULATORY ACTIVITY OF OENOTHEIN B

68. Ushio, Y., T. Fang, T. Okuda, and H. Abe. 1991. Modificational changes in 75. Baugh, J. A., and R. Bucala. 2001. Mechanisms for modulating TNF␣ in immune function and morphology of cultured macrophages by geraniin. Jpn. J. Pharma- and inflammatory disease. Curr. Opin. Drug Discov. Devel. 4: 635–650. col. 57: 187–196. 76. Brasier, A. R. 2006. The NF-␬B regulatory network. Cardiovasc. Toxicol. 6: 69. Murayama, T., N. Kishi, R. Koshiura, K. Takagi, T. Furukawa, and K. Miyamoto. 111–130. 1992. Agrimoniin, an antitumor tannin of Agrimonia pilosa Ledeb., induces in- 77. Chen, Y., L. Yang, and T. J. Lee. 2000. Oroxylin A inhibition of lipopolysac- terleukin-1. Anticancer Res. 12: 1471–1474. charide-induced iNOS and COX-2 gene expression via suppression of nuclear 70. Feldman, K. S., K. Sahasrabudhe, R. S. Smith, and W. J. Scheuchenzuber. 1999. factor-␬B activation. Biochem. Pharmacol. 59: 1445–1457. Immunostimulation by plant polyphenols: a relationship between tumor necrosis 78. Takechi, M., and Y. Tanaka. 1987. Binding of 1,2,3,4,6-pentagalloylglucose to factor-␣ production and tannin structure. Bioorg. Med. Chem. Lett. 9: 985–990. proteins, lipids, nucleic acids and sugars. Phytochemistry 26: 95–97. 71. Wang, C. C., L. G. Chen, and L. L. Yang. 2002. In vitro immunomodulatory effects of cuphiin D1 on human mononuclear cells. Anticancer Res. 22: 79. Kusuda, M., T. Hatano, and T. Yoshida. 2006. Water-soluble complexes formed 4233–4236. by natural polyphenols and bovine serum albumin: evidence from gel electro- 72. Apte, R. N., and E. Voronov. 2002. Interleukin-1: a major pleiotropic cytokine in phoresis. Biosci. Biotechnol. Biochem. 70: 152–160. tumor-host interactions. Semin. Cancer Biol. 12: 277–290. 80. He, Q., B. Shi, and K. Yao. 2006. Interactions of gallotannins with proteins, 73. Gabay, C. 2006. Interleukin-6 and chronic inflammation. Arthritis Res. Ther. amino acids, phospholipids and sugars. Food Chem. 95: 250–254. 8(Suppl 2): S3. 81. Teng, C. M., Y. F. Kang, Y. L. Chang, F. N. Ko, S. C. Yang, and F. L. Hsu. 1997. 74. Lejeune, F. J., D. Lienard, M. Matter, and C. Ruegg. 2006. Efficiency of recom- ADP-mimicking platelet aggregation caused by rugosin E, an ellagitannin iso- binant human TNF in human cancer therapy. Cancer Immun. 6: 6. lated from Rosa rugosa Thunb. Thromb. Haemost. 77: 555–561. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 Supplemental Material

for

Immunomodulatory Activity of Oenothein B Isolated

from Epilobium angustifolium

† Igor A. Schepetkin,* Liliya N. Kirpotina,* Andrei I. Khlebnikov, Larissa Jakiw,* Christie

L. Blaskovich,* Mark A. Jutila,* and Mark T. Quinn*

*Department of Veterinary Molecular Biology

Montana State University, Bozeman, MT 59717, USA

†Department of Chemistry

Altai State Technical University, Barnaul 656038, Russia Supplemental Table S1. NMR Spectroscopy of Subfraction S-3

1 H NMR [500 MHz, D2O]

-glucose:  4.47 (d, J 9 Hz, H-1), 5.09 (dd, J1 6.5, J2 9 Hz, H-2), 5.29 (t, J 9.5 Hz, H-3),

4.89 (t, J 10.5 Hz, H-4), 3.92 (dd, J1 5.5, J2 10.5 Hz, H-5), 4.45 (d, J 12.5 Hz, H-6), 3.75

(d, J 12.5 Hz, H-6); ring A: 6.41 (s, H-6); ring B: 6.28 (s, H-6΄); ring C: 6.53 (s, H-6΄); ring G: 6.95 (2H, s, H-2˝ and H-6˝); -glucose: 5.47 (d, J 3.4 Hz, H-1), 5.36 (dd, J1 3.4,

J2 10.5 Hz, H-2), 5.60 (t, J 10 Hz, H-3), 4.99 (t, J 9 Hz, H-4), 4.53 (dd, J1 7, J2 9 Hz, H-

5), 4.48 (d, J 12 Hz, H-6), 3.73 (d, J 12 Hz, H-6); ring A΄: 6.15 (s, H-6΄); ring B΄: 6.15

(s, H-6΄); ring C΄: 6.42 (s, H-6΄); ring G΄: 6.57 (2H, s, H-2˝ and H-6΄).

13 C NMR [400 MHz, D2O]

-glucose:  94.8 (C-1), 74.8 (C-2), 71.0 (C-3), 72.2 (C-4), 70.6 (C-5), 63.5 (C-6); ring

A: 124.6 (C-1΄), 116.3 (C-2΄), 143.5 (C-3΄), 136.1 (C-4΄), 145.8 (C-5΄), 106.5 (C-6΄),

169.8 (CO); ring B: 125.0 (C-1΄), 116.7 (C-2΄), 146.4 (C-3΄), 134.4 (C-4΄), 148.0 (C-5΄),

105.4 (C-6΄), 169.5 (CO); ring C: 115.0 (C-1΄), 139.7 (C-2΄), 140.5 (C-3΄), 140.8 (C-4΄),

142.9 (C-5΄), 113.7 (C-6΄), 168.8 (CO); ring G: 119.2 (C-1˝), 108.7 (C-2˝ and C-6˝),

144.3 (C-3˝ and C-5˝), 138.6 (C-4˝), 166.3 (CO); -glucose: 89.5 (C-1), 74.3 (C-2), 69.9

(C-3), 69.5 (C-4), 67.4 (C-5), 63.2 (C-6); ring A΄: 121.4 (C-1΄), 115.1 (C-2΄), 144.0 (C-

3΄), 134.8 (C-4΄), 144.8 (C-5΄), 106.9 (C-6΄), 169.1 (CO); ring B΄: 124.8 (C-1΄), 116.7

(C-2΄), 144.3 (C-3΄), 135.9 (C-4΄), 147.5 (C-5΄), 106.5 (C-6΄), 169.6 (CO); ring C΄: 114.3

(C-1΄), 137.5 (C-2΄), 139.1 (C-3΄), 141.6 (C-4΄), 142.3 (C-5΄), 109.8 (C-6΄), 167.1 (CO); ring G΄: 117.7 (C-1˝), 110.3 (C-2˝ and C-6˝), 144.4 (C-3˝ and C-5˝), 138.6 (C-4˝), 169.4

(CO). Supplemental Figure S1. Chemical Structure of Oenothein B

OH -glucose HO 5' A OH HO 3' 1' O 5' OH O 5 O HO 3' 1' 1 OH O O C' B 3 O O 1' 3' OH O OH HO 5' O G O O OH OH O OH HO B' O OH O C 1 OH 5 O HO O OH 3 O OH O A' O O OH HO 5" 1" OH G' 3" -glucose HO OH Supplemental Figure S2. Mass Spectrum of Subfraction S-3

(M+2Na)2+

(M+Na)+

(2M+3Na)3+

Mass spectrometry experiments were performed using a Bruker Microtof high resolution time of flight mass spectrometer (Bruker Daltonics, Inc., Billerica, MA). Supplemental Figure S3. Structures of Related Compounds Under Investigation

OH OH OH HO OH HO OH OH O OH Pyrogallol Pyrocatechol Gallic acid

OH OH HO O OH OH HO O HO OHO O O OH O O HO 3,4-dihydroxybenzoic HO O OH acid (protocatechuic acid) HO Corilagin OH

OH HO OH

OH OH HO O OH O O HO O O O OH O O HO O O O HO

OH HO OH OH

1,2,3,4,6-pentakis-O-galloyl--D-glucose (PGG) >>=?$! =>/@?E

!%

!$

!# &/&%<<=

!

VW=/X#W< /YZ[Z&#$ZW  >\ \ >\ ]\$^&_ X[` <  ` #<  X[ > Z ^=E X[ ! j >/>/ / Z/ _ />>= />! V >/ / =!! >/=>/ = ^>= / >/E > ^>=/!///Xqqq!W[E//& /!