The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
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Production of Nitric Oxide in Raw 264.7 Macrophages treated with Ganoderan, the ${\beta}-Glucan$ of Ganoderma lucidum

영지의 균사체성 ${\beta}-glucan$에 의한 Raw 264.7 대식세포의 Nitric Oxide생성

  • Published : 1998.06.30
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Abstract

Ganoderan (GAN), an immunomodulating ${\beta}-glucan$ of G. lucidum, induces potent antitumor immunity in tumor-bearing mice. This study was set up to elucidate the ability of macrophage activation of GANs. GAN-treated Raw 264.7 macrophages showed enhanced production of nitric oxide (NO). The ability of GANs to produce NO was based on differences in chemical composition of GANs obtained from the mycelium on various carbon sources and mycelial fractionation. The highest NO production was observed in CW-AS-WS polysaccharide which was extracted from the mycelial wall. GAN-treated Raw 264.7 cells gave a 2-to 5-fold (24 hr) formation of NO levels compared with those treated with medium only. Partial removal of the protein in the extracellular GAN by TCA treatment did appreciably reduce its capacity to secrete NO. The mixture effect of GAN and LPS increased the nitric oxide secretion from RAW 264.7. The cell proliferation of GAN-treated Raw 264.7 cell tines inhibited as compared with its control. Of the culture supernatant of macrophage activated by GAN, the percentage of cytotoxicity against mouse leukemia L1210 cells was slightly dependent on the amount of NO in the culture supernatants of the activated-macrophages. These results indicate that the ${\beta}-glucan-related$ polysaccharides of the higher fungus activate macrophage and release nitric oxide. It also suggests that murine macrophages possess certain receptors for ${\beta}-anomeric$ glucans and play a critical role of ${\beta}-glucan-related$ tumor killing mechanism.

영지균사체로부터 기능적인 면역활성 물질을 얻고자, 여러 조건으로 추출된 ${\beta}-glucan$성 다당류(GAM)의 Raw 264.7 대식세포 활성 정도를 알아보았다. GAN으로 자극된 Raw 264.7 대식세포는 대조군보다 약 $2{\sim}5$배의 nitric oxide 생성을 촉진하였다. 대식세포의 활성화 정도는 GAM의 구조와 분자량 그리고 화학적 조성에 따라 NO생성에 차이를 보였다. 서당을 탄소원으로 배양된 균사체로부터 추출된 알칼리 가용성이며 수용성인 G-BUC로 활성화된 Raw 264.7세포는 $22{\mu}M$의 NO생성을 유도하였다. G-SUC(WS)는 87%의 탄수화물과 3.4%의 단백질로된 평균분자량 30 kD의 다당류이다. 탄수화물의 구성당은 48%의 포도당과 22%의 mannose등으로 구성된 glucomannan성 GAM이다. 세포분획성 GAM가운데 세포벽으로부터 추출된 알칼리 가용성(CW-AB-WS)인 ${\beta}-glucan$성 다당류가 가장 많은 NO생성을 촉진시켜 영지의 대식세포 활성 다당류로 가장 잠재력이 있는 다당류인 것으로 나타났다. NO생성에 GAN과 대식세포 활성물질(LPS 및 $IFN-{\gamma}$)의 혼합 효과는 독자적인 자극보다는 혼합하여 자극했을 때 NO의 생성이 증폭되었다. GAM으로 활성화된 대식세포의 유사분열은 대조군에 비해 현저히 억제되었으며, 활성화된 대식세포의 배양액은 in vitro에서 L1210 암세포 주에 세포독성을 나타내었다. 이와 같은 결과로 보아 영지의 ${\beta}-glucan$성 다당류(GAN)는 ${\beta}-anomeric$ glucan receptor를 갖고 있는 대식세포를 자극하여 NO를 생성하며, 이때 NO생성의 촉진하는 다당류의 조건은 어느정도의 수용성을 갖고 있는 소량의 단백질이 포함된 고분자성 ${\beta}$glucan다당류이다.

Keywords

References

  1. J. Immunol. v.126 Evidence for a multistep mechanism of cytolysis by Gactivated macrophages: the interrelationship between the capacity for cytolysis, target binding, and secretion of cytolytic factor Adams, D.O.;Marino, P.A.
  2. Proc. Natl. had. Sci. USA v.72 An endotoxin-induced serum factor that causes necrosis of tumors Carswell, E.A.;Old, L.J.;Kassel, R.L.;Green, S.;Fiore, N.;Williamson, B.
  3. Nature v.222 Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes Chihara, G.;Maeda, Y.;Hamuro, T.;Sasaki, T.;Fukuoda, F.
  4. J. Exp. Med. v.175 Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages Chan, J.;Xing, Y.;Maglliozzo, R.S.;Bloom, B.R.
  5. Eur. J. Immunol. v.23 Repeated induction of nitric oxide synthetase and leishmanicidal activity in murine macrophage Chun, Q.C.;Assreuy, J.;Xu, D.;Charles, I.;Liew, F.Y.;Moncada, S.
  6. Cancer Res. v.54 Activated murine macrophage induce apotosis in tumor cells through nitric oxide-dependent or independent mechanism Cui, S.;Jonathan, S.Reichner;Romeo, B.Mateo;Albina, Jorge E.
  7. Immunology v.81 In vitro human lymphocyte proliferative responses to a glycoprotein of the yeast Saccharomyces cerevisiae Darroch, C.J.;Christmas, S.E.;Barnes, R.M.R.
  8. J. Gen. Microbiol. v.76 Release of protoplasts from Schizophyllum commune by combined action of purified ${\alpa}$-1,3glucanase and chitinase derived from Trichoderma viride De Vries, O.M.H.;Wessels, J.G.H.
  9. J. Immunol. v.144 Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production Ding, A.J.;Nathan, C.F.;Stuehr, D.J.
  10. Planta Medica v.55 Polysaccharides in pharmacy: Current applications and future concepts Franz, G.
  11. Exp. Cell Res. v.174 Characteristics of the ${\beta}$-glucan receptor of murine macrophage Goldman, R.
  12. Biochemical J. v.219 Oxygen toxicity, oxygen radicals, transition metals and disease Halliwell, B.;Cutteridge, J.M.C.
  13. Korean J. Mycol. v.23 The effects of carbon sources on antitumor and anticomplementary activities of Ganoderan extracted from the mycelium of Ganoderma lucidum IY009 Han, M.D.;Lee, J.W.;Jeong, H.;Chung, S.K.;Lee, S.Y.;Yoon, K.H.
  14. Korean J. Mycol. v.23 The composition and bioactivities of Ganoderan by mycelial fractionation of Ganoderma lucidum IY009 Han, M.D.;Jeong, H.;Lee, J.W.;Back, S.J.;Kim, S.U.;Yoon, K.H.
  15. Proc. Soc. expo Biol.(N. Y) v.139 In vitro nonimmunological destruction of cells with abnormal growth characteristics by adjuvant activated macrophages Hibbs, J.B.;Lambert, L.H.;Remington, J.S.
  16. J. Immunol. v.138 L-Arginine is required for expression of the activated effector mechanism causing selective metabolic inhibition in target cells Hibbs, J.B.;Vavrin, Z. Jr.;Tainer, R.R.
  17. J. Immunol. v.144 Cytolytic mechanisms of activated macrophages; Tumor necrosis factor and L-Arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages Higuchi, M.;Higagashi, N.;Taki, H.;Osawa, T.
  18. J. Immunol. v.141 Tumor necrosis factor and IL-1 associated with plasma membrane of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both Ichinose, Y.;Bakouche, O.;Tsao, J.Y.;Fildler, I.J.
  19. Contemp. Top. Immunobiol. v.14 Activation of macrophages for tumor cyto-toxicity Johnson, W.J.;Somers, S.D.;Adams, D.O.
  20. Gann. v.60 Host mediated antitumor action of Schizophyllum commune Komatsu, N.;Okubo, S.;Kikumoto, S.;Kimura, K.;Saito, G.;Sasaki, S.
  21. J. Immunol. v.141 Specific endotoxic lipopolysaccharide-binding sites on splenocytes and splenocyte subpopulations Lei, M.G.;Morrison, D.C.
  22. Cell. Immunol. v.77 Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas Loewenstein, J.;Rottem, S.;Gallily, R.
  23. J. Biol. Chem. v.268 Nitric oxide synthetase structure and mechanism Marletta, M.A.
  24. J. Immunol. v.127 Macrophage activation for tumor cytotoxicity: characterization of priming and trigger signal during lymphokine activation Meltzer, M.S.
  25. New Engl. J. Med. v.329 The arginine-nitric oxide pathway Moncada;Higgs, A.
  26. J. Immunol. Methods. v.65 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays Mosmann, T.
  27. J. Immunol. v.114 Macrophages activated in vitro with lymphocyte mediators kill neoplastic but not normal cells Pissens, W.F.;Churchill, W.H.;David, J.R.
  28. Immunology Roitt, I.;Brostoff, J.;Male, D.
  29. Complement v.4 Specific of membrane complement receptor type three ($CR_3$) for ${\beta}$-glucan Ross, G.D.;Cain, J.A.;Myones, B.L.;Newman, S.L.;Lachmann, P.J.
  30. Cell. Immunol. v.35 Functional and morphological characteristics of interferon-treated macrophage Schultz, R.M.;Chirigos, M.A.;Heine, U.I.
  31. Kor. J. Pharmacogn v.16 Studies on inorganic composition and immunopotentiating activity of Ganoderma lucidum in Korea Shin, H.W.;Kim, H.W.;Choi, E.C.;Kim, B.K.
  32. J. Immunol. v.131 The binding of tumor cells by murine mononuclear phagocytes can be devided into two, qualitatively distinct types Somers, S.D.;Mastin, J.P.;Adams, D.O.
  33. J. Immunol. v.144 IL-1 secretion by macrophages. Enhancement of IL-1 secretion and processing by calcium inophores Suttles, J.;Giri, J.G.;Mizel, S.B.
  34. Gann v.65 Proteinbound polysaccharide preparation, PS-K, effective against sarcoma-180 and rat ascites hepatoma AH-13 by oral use Tsugagoshi, S.;Ohashi, F.