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