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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Studies of Xanthium strumarium Extract Suppressing Th17-cell Differentiation and Anti-dermatitic Effect in BMAC-induced Atopy Dermatitis of NC/Nga Mice

창이자 추출물이 아토피 피부염 유발 생쥐의 비장 세포 Th17의 세포분화 억제에 따른 아토피 피부 상태에 미치는 영향

  • Kim, Kum-Lan (Department of Bioengineering Graduate School at Konkuk University) ;
  • Choe, Tae-Boo (Department of Bioengineering Graduate School at Konkuk University)
  • 김금란 (건국대학교 대학원 생물공학과) ;
  • 최태부 (건국대학교 대학원 생물공학과)
  • Published : 2009.08.29
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Abstract

Xanthii fructus which is well known as "Chang-ihjah" in Korea is the dried fruit of Xanthium strumarium L. (or Xanthium sibiricum PATR. Ex WIDD., Asteraceae. XS). Water extract of this fruit has been used for treatment of various inflammatory diseases such as tympanitis, allergic rhinitis, or ozena as alternative therapy material usually by oral administration in far Eastern countries including Korea. In this study, the effect of XS extract (XS-E) or XS-30% acetone fraction layer (XS-30% AFL) on the differentiation of $CD4^+$ T cells isolated from NC/Nga mouse and the production of IL-17 was investigated. The experimental results showed that $100\;{\mu}g$/mL of XS-E could decrease the production of IL-17 by $CD4^+$ Th17 cells by 2 fold and only $20\;{\mu}g$/mL of XS-30% AFL could inhibit 3.5 fold. The amount of IL-17A and IL-22 mRNA determined by real-time PCR was decreased remarkably when XS-E or XS-30% AFL was treated on $CD4^+$ Th17 cells(p<0.01, p<0.001). The amount of IL-17A protein determined by ELISA was also decreased remarkably(p<0.05, p<0.001). To study the effect of XS-E or XS-30% AFL on the proliferation of Th17 cells, $CD4^+$ T cells of a NC/Nga mouse was firstly differentiated by rIL-6/TGF-$\beta$ and then stimulated by rIL-23. The control group of Th17 cells were doubled every each day, while those of XS-E or XS-30% AFL treated group were shown to be delayed remarkably by these extracts. In conclusion, XS can inhibit the differentiation of Th17 cells of NC/Nga mouse and the production of IL-17 successfully, which may be a beneficial result for the treatment of atopic skin dermatitis.

아토피피부염 (AD)은 천식, 음식 알레르기, 비염 같은 전신아토피질환을 동반하는 만성재발성 피부염증질환이다. 아토피피부염과 관련된 IL-17의 임상적 역할은 다양한 조건에서 보고되고 있으며, 또한 건선 피부 상태에 깊숙이 관여하고 있다. IL-17은 각질세포 (keratinocytes)에서 과잉으로 생산되며, 아토피피부염의 말초임파구에서도 다량 생성됨을 세포내염색을 통하여 확인된 바 있다. 본 연구에서는 창이자 추출물 (XS-E와 XS-FL)이 NC/Nga 생쥐의 $CD4^+$ T 세포에서 유도된 Th17 세포의 분화억제 및 IL-17의 생산량 감소 효과에 대한 실험을 하였다. 그 결과 XS-E와 XS-FL을 처리한 섬유아세포에서 세포독성은 나타나지 않았고, 4일간 XS-E와 XS-FL에 동시배양 한 $CD4^+$ T 세포의 IL-17 생산량을 FACS로 분석한 결과 $100\;{\mu}g$/mL XS-E 처리군의 IL-17 생산량은 32.3%로 대조군에 비하여 2배 이상의 감소를 나타내었으며, $20\;{\mu}g$/mL XS-30% AFL (acetone XS-FL) 처리군의 Th17 세포는 19.6%으로 대조군에 비하여 3.5배 억제되었다. 또한 real-time PCR을 이용하여 IL-17A와 IL-22 mRNA의 유전자 발현량을 비교 분석한 결과, IL-17A와 IL-22 mRNA의 유전자발현의 RQ값은 XS-E와 XS-30% AFL를 처리한 실험군이 대조군에 비하여 유의성 있는 감소를 나타내었다(p < 0.01, p < 0.001). ELISA로 측정한 IL-17A 생산량은 XS-E와 XS-30% AFL를 처리한 실험군이 대조군에 비하여 현저히 감소 (p < 0.05, p < 0.001)하였다. Th17 세포의 증식을 알아보기 위하여, rIL-6와 TGF-$\beta$로 분화시킨 Th17 세포를 CFSE로 표지한 후 rIL-23 처리를 하여 4일간 배양하여 증식을 유도시켰다. 대조군의 Th17 세포 분열은4일 동안 4번에 걸쳐 비슷한 세포수의 증식이 일어나는 것을 CFSE를 통하여 확인하였고, XS-30% AFL 처리군은 CFSE의 형광 분포가 점점 감소하여 Th17 세포의 증식이 억제됨을 알 수 있었다.

Keywords

References

  1. Johansson, S. G., L. O. Cardell, T. Foucard, P. Montan, P. Odeback, and M. Palmqvist (2006), Revised, global nomenclature for allergy. Unambiguous terms create clarity and prevent misunderstandings, Lakartidningen 8-14, 103, 379-383
  2. Ma, D., X. Zhu, P. Zhao, C. Zhao, X. Li, and Y. Zhu (2008), Profile of Th 17 cytokines (lL-17, TGF -beta, IL-6) and Th1 cytokine (lFN-gamma) in patients with immune thrombo cytopenic purpura, Ann Hemato 87, 899-904 https://doi.org/10.1007/s00277-008-0535-3
  3. Nograles, K. E., L. C. Zaba, E. Guttman-Yassky, J. Fuentes-Duculan, and M. Suarez-Farinas (2008), Th17 cytokines interleukin (IL )-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways. Br. J. Dermatol. 159, 1092-1102
  4. Tesmer, L. A., S. K. Lundy, S. Sarkar, and D. A. Fox (2008), Th17 cells in human disease. lmmunol. 223, 87-113 https://doi.org/10.1111/j.1600-065X.2008.00628.x
  5. Zelante, T., A. De Luca, C. D'Angelo, S. Moretti, and L. Romani (2009), IL-17/Th17 in anti-fungal immunity : what's new? Eur Jlmmunol. 39, 645-648 https://doi.org/10.1002/eji.200839102
  6. Infante-Duarte, C., H. F. Horton, M. C. Byme, and T. Kamradt (2000), Microbial lipopeptides induce the production of IL-17 in Th cells. J. lmmunol. 165, 6107-6115
  7. Langrish, C. L., Y. Chen, W. M. Blumenschein, J. Mattson, B. Basham， and J. D. Sedgwick (2005), IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J. Exp. Med. 201, 233-240 https://doi.org/10.1084/jem.20041257
  8. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Tumer, T. L. Murphy, and K. M. Murphy (2005), Interleukin 17 -producing CD4+ effector T cells develop via alineage distinct from the T helper type 1 and 2 lineages. Nat. lmmunol. 6, 1123-1132 https://doi.org/10.1038/ni1254
  9. McAllister, F., A. Henry, J. L. Kreindler, P. J. Dubin, and L. Ulrich (2005), Role of IL-17 A, IL-17F, and the IL-17 rece:ptor in regulating growth-related oncogene-alpha and granulocyte colony-stimulating factor in bronchial epithelium : implications for airway inflammation in cystic fibrosis, J. lmmunol. 175, 404-412
  10. Conte, F. P., C. Barja-Fidalgo, W. A. Verri, F. Q. Cunha, G. A. Rae, and C. Penido (2008), Endo the lins modulate inflammatory reaction in zymosan-induce darthritis : participation of L TB4, TNF -alpha, and CXCL-l, J. Leukoc. Biol. 84, 652-660 https://doi.org/10.1189/jlb.1207827
  11. Kleinewietfeld, M., F. Puentes, G. Borsellino, L. Battistini, O. Rotzschke, and K. Falk (2005), CCR6 expression defines regulatory effector/memory-like cells within the CD25(+)CD4+ T-cell subset, Blood. 105, 2877-2886 https://doi.org/10.1182/blood-2004-07-2505
  12. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, and Y. H. Wang (2005), A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17, Natlmmunol. 6, 1133-1141 https://doi.org/10.1038/ni1261
  13. Manel, N., D. Unutmaz, and D. R. Littman (2008), The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor ROR gammat, Nat. lmmunol. 9, 641-649 https://doi.org/10.1038/ni.1610
  14. Zhou, L., J. E. Lopes, M. M. Chong, II. Ivanov, R. Min, and G. D. Victora (2008), TGF-beta-induced Foxp 3 inhoibits T(H) 17 cell differentiation by antagonizing ROR gammatfunction, Nature 453, 236-240 https://doi.org/10.1038/nature06878
  15. Cookson, W. (1999), The alliance of genes and environment in asthma and allergy, Nature 402, 5-11
  16. Zhan, Y., S. Gεrondakis, E. Coghill, D. Bourges, Y. Xu, and J. L. Brady (2008), Glucocorticoid-induced TNF receptor exprεssion by T cells is recipro cally regulated by NF-kappa B and NFA T, J. lmmunol. 181, 5405-5413
  17. Bames, P. J. (1999), Therapeutic strategies for allergic diseases， Nature 402, 31-38
  18. Leung, D. Y. (2000), Atopic dermatitis : new insights and opportunities for therapeutic intervention, J. Allergy Clin lmmunol. 105, 860-876 https://doi.org/10.1067/mai.2000.106484
  19. Novak, N., T. Bieber, and D. Y. Leung (2003), Immune mechanisms leading to atopic dεrmatitis， J. Allergy Clin lmmunol. 112, 128-139 https://doi.org/10.1016/j.jaci.2003.09.032
  20. Kim, H. M., J. M. Yi, and K S. Lim (1999), Magnoliae flos inhibits mast cell-dependent immediate-type allergic reactions ， Pharmacol. Res. 39, 107-111 https://doi.org/10.1006/phrs.1998.0414
  21. Koibuchi, Y., A. Ichikawa, M. Nakagawa, and K. Tomita (1985), Histamine release induced from mast cell s by active components of compound 48/80, Eur J. Pharmacol. 115, 163-170 https://doi.org/10.1016/0014-2999(85)90687-9
  22. Chu, T. R. and Y. C. Wei (1965), Studies on the principal unsaturated fatty acids of the seed oil of Xanthium strumarium L, Yao. Xue Xue Bao 12, 709-712
  23. Baltzly, R. and J. S. Buck (1949), A family of longacting depressors ，J. Am. Chem. Soc. 71, 1301-1305 https://doi.org/10.1021/ja01172a045
  24. Hsu, F. L., Y. C. Chen， and J. T. Cheng (2000), Caffeic acid as active principle from the fruit of Xanthium strumarium to lower plasma glucose in diabetic rats, Planta Med. 66, 228-230 https://doi.org/10.1055/s-2000-8561
  25. Segal, D. M., J. D. Taurog, and H. Metzger (1977), Dimeric immunoglobulin E serves as a unit signal for mast cell degranulation, Proc. Natl. Acad. Sci U.S.A. 74, 2993-2997 https://doi.org/10.1073/pnas.74.7.2993
  26. Radunz, H. E. (1988), Screening in drug design-modem methods in active-substance finding, Pharm Unserer Zeit. 17, 161-176 https://doi.org/10.1002/pauz.19880170602
  27. Kim, Y. S., J. S. Kim, S. H. Park, S. U. Choi, C. O. Lee, and Kim, S. K. (2003), Two cytotoxics esquiter penel actones from the leaves of Xanthium strumarium and their in vitro inhibitory activity on famesyl trans ferase, Planta Med. 69, 375-377 https://doi.org/10.1055/s-2003-38879
  28. Vestergaard, C., H. Y oneyama, and K. Matsushima (2000), The NC/Nga mouse : a model for atopic dermatitis， Mol. Med. Todary 6, 209-210 https://doi.org/10.1016/S1357-4310(00)01683-X
  29. Tohda, C., H. Sugahara, Y. Kuraishi, and K Komatsu (2000), Inhibitory eftìect of Byakko-ka-ninjin-to on itch in a mouse model of atopic dermatitis， Phytother Res. 14, 192-194 https://doi.org/10.1002/(SICI)1099-1573(200005)14:3<192::AID-PTR609>3.0.CO;2-F
  30. Maric, D. and J. L. Barker (2004), Neural stem cells redefined : a F ACS perspεctive， Mol. Neurobiol. 30, 49-76 https://doi.org/10.1385/MN:30:1:049
  31. Klein, D., B. Bugl, W. H. Gunzburg, and B. Salmons (2000), Accurate estimation of transduction efficiency necessitates a multiplex real-time PCR, Gene Ther. 7, 458-463 https://doi.org/10.1038/sj.gt.3301112
  32. Steuerwald, N., J. Cohen, R. J. Herrera, and C. A. Brenner (2000), Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR, Mol. Hum Reprod. 6, 448-453 https://doi.org/10.1093/molehr/6.5.448
  33. Liang, S. C., X. Y. Tan, D. P. Luxenberg, R. Karim, K. Dunussi-Joannopoulos, and M. Collins (2006), Interleukin(IL)-22 and IL-17 a recoexprle ssed by Th17 cells and cooperatively enhance expression of antirnicrobial peptides, J. Exp. Med. 203, 2271-2279 https://doi.org/10.1084/jem.20061308
  34. Thone, F., B. Schwanhausser， D. Becker, M. Ballmaier, and D. Bumann, D, (2007), FACS-isolation of Salmonellainfected cells with defined bacterial load from mouse spleen, J. Microbiol Methods 71, 220-224 https://doi.org/10.1016/j.mimet.2007.08.016
  35. Ghosh, S., S. Gepstein, J. J. Heikkila， and E. B. Dumbroff (1988), Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues， Anal Biochem. 169, 227-233 https://doi.org/10.1016/0003-2697(88)90278-3
  36. Carson, S. D. and P. G. Archer (1986), Tissue factor activity in HeLa cells measured with a continuous chromogenic assay and ELISA reader, ThrombRes.15; 41, 185-195
  37. Korda, A., Z. Wrobel， and K. Gwardiak (2006), An efficient route from trifluoroacetates to water soluble free amines using Diaion HP-20, Amino Acids 30, 95-98 https://doi.org/10.1007/s00726-005-0228-3
  38. Soylak, M., F. Armagan, L. Elci, and M. Dogan (2001), A new preconcentration and separation method for flame atomic absorption spectrometric determinations of some trace metal ions on a Diaion HP-20 colunm, Ann Chim. 91, 637-647
  39. Vichai, V. and K. Kirtikara (2006), Sulforhodamine B colorimetric assay for cytotoxicity screening, Nat. Protoc. 1, 1112-1116 https://doi.org/10.1038/nprot.2006.179
  40. Suto, H., H. Matsuda， K Mitsuishi, K. Hira, T. Uchida, and T. Unno (1999), NC/Nga mice : a mousε model for atopic dermatitis, lnt. Arch. Allergy. lmmunol. 120, 70-75 https://doi.org/10.1159/000053599
  41. Lee, J. B., J. M. Kim, S. J. Kim, J. H. Park, S. H. Hong, and S. I. Roh (2005), Comparative characteristics of three human εmbryonic stem cell lines, Mol. Cells. 19, 31-38
  42. Kobayashi, T., S. Okamoto, T. Hisamatsu, N. Kamada， H. Chinen, and R. Saito (2008), IL23 diftìerent I ally regulates the ThllTh17 balance in ulcerative colitis and Crohn’s disease ， Gut 57, 1682-1689 https://doi.org/10.1136/gut.2007.135053
  43. Baecher-Allan, C. M., and D. A. Hafler (2005), Functional analysis of highly defined， FACS-isolated populations of human regulatory CD4+CD25+T cells,Clin. lmmunol. 117, 192-193
  44. Hadjantonakis, A. K and A. Nagy (2000), FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 27, 95-98 https://doi.org/10.1002/1526-968X(200007)27:3<95::AID-GENE10>3.0.CO;2-A
  45. Chevrel， G., P. Gamero, and P. Miossec (2002), Addition of interleukin 1 (lL1) and IL17 soluble receptors to a tumour necrosis factor alpha soluble receptor more effiectively reduces the production of IL6 and macrophage inhibitory protein-3alpha and increases that of collagen in an in vitro model of rheumatoid synoviocyte activation, Ann Rheum Dis. 61, 730-733 https://doi.org/10.1136/ard.61.8.730
  46. Housseau, F., K. R. Lindsey, S. D. Oberholtzer, M. I. Gonzales, P. Boutin, and A. K. Moorthy (2002), Quantitative real-time RT -PCR asam ethod for monitoring T lymphocyte reactivityt of ull-length tyrosinase protein in vaccinated melanoma patients, J. lmmunol. Methods 266, 87-103 https://doi.org/10.1016/S0022-1759(02)00104-7
  47. Lyons, A. B. (2000), Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution, J. lmmunol. Methods 243, 147-154 https://doi.org/10.1016/S0022-1759(00)00231-3
  48. Godfrey, W. R., M. R. Krampf, P. A. Taylor, and B. R. Blazar (2004), Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation, Bloodl. 103, 1158-1165 https://doi.org/10.1182/blood-2003-04-1098
  49. Martin, A. and M. Clynes (1993), Comparison of 5 microplatε colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays, Cytotechnology. 11, 49-58 https://doi.org/10.1007/BF00749057
  50. Gurbay, A., C. Garrel, M. Osman, M. J. Richard, A. Favier, and F. Hincal (2002), Cytotoxicity in ciprofloxacin-treated human fibroblast cells and protection by vitanlin E, Hum. Exp. Toxicol. 21, 635-641 https://doi.org/10.1191/0960327102ht305oa
  51. McKenzie， B. S., R. A. Kastelein, and D. J. Cua (2006), Understanding the IL-23-IL-17 immune pathway, Trends lmmunol. 27, 17-23 https://doi.org/10.1016/j.it.2005.10.003
  52. Hunter, C. A. (2005), New IL-12-family members : IL-23 and IL-27, cytokines with divergent functions, Nat. Rev. lmmunol. 5, 521-531 https://doi.org/10.1038/nri1648
  53. Mangan, P. R., L. E. Harrington, D. B. 0 ’Quin, W. S. Helms, D. C. Bullard, and C. O. Elson (2006 May), Transforming growth factor-beta induces development of the T(H)17 lineage, Nature 441, 231-234 https://doi.org/10.1038/nature04754
  54. Chen, Z., A. Laurence, Y. Kanno, M. Pacher-Zavisin, B. M. Zhu, and C. Tato (2006), Selective regulatory function of Socs 3 intheformation of IL-17 secreting T cells, Proc. Natl. Acad. Sci. USA. 103, 8137-8142 https://doi.org/10.1073/pnas.0600666103
  55. Hoeve, M. A., N. D. Savage, T. de Boer, D. M., de, Langenberg, Waal. R. Malefyt, and T. H. Ottenhoff (2006), Divergent effects of IL-12 and IL-23 on the production of IL-17 by hUlllan T cells. Eur, J. lmmunol. 36, 661-670 https://doi.org/10.1002/eji.200535239
  56. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger (2006), TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17 -producing T cells, lmmunity 24, 179-189
  57. Bettelli, E., Y. Carrier, W. Gao, T. Kom, T. B. Strom, and M. Oukka (2006), Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells, Nature 441, 235-238 https://doi.org/10.1038/nature04753
  58. Kolls, J. K and A. Linden (2004), Interleukin-17 family members and inf1ammation, lmmunity 21, 67-476
  59. Kom, T., M. Oukka, V. Kuchroo, and E. Bettelli (2007), Th17 cells : effector T cells with inflammatory properties, Semin. lmmunol. 19, 362-371 https://doi.org/10.1016/j.smim.2007.10.007
  60. Lubberts, E., P. Schwarzenberger, W. Huang, J. R. Schurr, J. J. Peschon, and W. B. van den Berg (2005), Requirement of IL-17 receptor signaling in radiationresistant cells in the joint for full progression of destructivesy no vitis, J. lmmunol. 175, 3360-3368
  61. Chabaud, M. and P. Miossec (2001), The combination of tumor necrosis factor alpha blockade with interleukin-1 and interleukin-17 blockade is more εffective for controlling synovial inflammation and bone resorption in an ex vivo model, Arthritis Rheum 44, 1293-1303 https://doi.org/10.1002/1529-0131(200106)44:6<1293::AID-ART221>3.0.CO;2-T
  62. Yen, D., J. Cheung, H. Scheerens, F. Poulet, T. McClanahan, and B. McKenzie (2006), IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6, J. Clin. Invest. 116, 1310-1316 https://doi.org/10.1172/JCI21404
  63. Chabaud, M., J. M. Durand, N. Buchs, F. Fossiez, G. Page, and L. Frappart (1999), HUlllan interleukin-17 : A T cell-derived proinflalllillatory cytokine produced by the rheumatoid synovium, Arthritis Rheum 42, 963-970 https://doi.org/10.1002/1529-0131(199905)42:5<963::AID-ANR15>3.0.CO;2-E
  64. Joosten, L. A., T. R. Radstake, E. Lubberts, L. A. Bersselaar, P. L. van Riel, and P. L. van Lent (2003), Association of interleukin-18 expression with enhance dlevels of both interleukin-1 beta and tumor necrosis factor alpha in kneesy no vial tissue of patients withr heumat oidarthritis, Arthritis Rheum 48, 339-347 https://doi.org/10.1002/art.10814
  65. Fujino, S., A. Andoh, S. Bamba, A. Ogawa, K Hata, and Y. Araki (2003), Increased expression of interleukin 17 in inflammatory bowel disease, Gut. 52, 65-70 https://doi.org/10.1136/gut.52.1.65