舞茸提取物Dfraction能增强NK细胞的细胞毒性作用.docx

1、舞茸提取物Dfraction能增强NK细胞的细胞毒性作用声明：本文摘自Oncology Reports，由希腊知名的科技出版社Spandidos Publications出版发行，创刊于1994年，月刊。该杂志内容覆盖肿瘤领域广泛的基础和应用研究，注重于发表肿瘤发生、转移和流行病学方面的原创性论文和综述。为方便广大读者阅读，我们对论文摘要进行了中文翻译，译文仅供参考，如有不足之处，敬请指正。-本文摘自舞茸D-fraction官方网站，转载请注明【参考译文】 Enhancement of cytotocicity of NK cells by D-Fraction, a polysacchari

2、de from Grifola frondosa舞茸提取物D-fraction能增强NK细胞的细胞毒性作用 摘 要 在先天免疫中，在不受MHC抗原限制的情况下被激活的自然杀伤（NK）细胞能攻击和消灭病原体如细菌、病毒。有报道称被IL-12激活的NK细胞在穿孔蛋白介导的细胞凋亡中能识别和杀死肿瘤细胞。我们曾报道，一种从舞茸中提取的多糖D-fraction能激活巨噬细胞、树突细胞以及T细胞，并能抑制肿瘤生长。尽管如此，舞茸D-fraction 在先天免疫反应中对NK细胞功能的影响并不清楚。在此项试验中，MM-46乳腺癌荷瘤小鼠连续三天腹腔内注射舞茸D-fraction，从而研究其对NK细胞活性和细

3、胞毒性的作用。结果显示D-fraction能显著增强NK细胞对于NK敏感性靶细胞YAC-1细胞的细胞毒性以及其CD223的表达量。D-fraction也能增加巨噬细胞的CD86表达量。另外，与对照组相比，脾细胞培养上清液以及血清中的IL-12量也增加，相应地NK细胞中IL-12受体1的表达量也增加。这些结果表明D-fraction通过激活巨噬细胞分泌IL-12来增强NK细胞的细胞毒性。关键词：NK细胞，细胞毒性，白介素-12受体论文原文 ONCOLOGY REPORTS 13: 497-502, 2005 Enhancement of cytotoxicity of NK cells by D

4、-Fraction, a polysaccharide from Grifola frondosaNORIKO KODAMA1, AKIHIRO ASAKAWA2, AKIO INUI2, YUKI MASUDA and HIROAKI NANBA11Department of Microbial Chemistry, Kobe Pharmaceutical University, 4-19-1, Motoyama- kitamachi, Higashinada-ku, Kobe 658-8558; 2Division of Diabetes, Digestive and Kidney Dis

5、eases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Received August 10, 2004; Accepted October 4, 2004Abstract.In innate immunity, activated natural killer (NK) cells attack and damage pathogens such as bact

6、eria and virus without restriction by the MHC antigen. NK cells activated by IL-12 have been reported to recognize and kill tumor cells in perforin-mediated apoptosis. We have reported that D-Fraction, a polysaccharide extracted from the maitake mushroom (Grifola frondosa), activates macrophages, de

7、ndritic cells, and T cells and inhibits the growth of tumor cells. However, the effects of D-Fraction on NK cell function in the innate immune response are not well known. In the present study, we administered D-Fraction to MM-46 mammary tumor-bearing C3H/HeJ mice intraperitoneally for 3 consecutive

8、 days and investigated its effects on the activation and cytotoxicity of NK cells. D-Fraction significantly enhanced the cytotoxicity against NK-sensitive YAC-1 cells and the expression of CD223 on NK cells. D-Fraction also increased the expression of CD86 on macrophages. In addition, the levels of

9、IL-12 in the culture supernatant of whole spleen cells and in serum increased, compared with the control corresponding to an increase in expression of IL-12 receptor BI on NK cells. These results suggest that D-Fraction enhances the cytotoxicity of NK cells through the production of IL-12 by macroph

10、ages activated by D-Fraction.Key words: natural killer cell, cytotoxicity, interleukin-12 receptorIntroductionNatural killer (NK) cells are responsible for innate immunity and have the ability to recognize and kill tumor cells without completely expressing histocompatibility complex (MHC) class I an

11、d II antigens, by which NK cells are distinguished from B and T cells (1). Despite some similarities to T cells, NK cells are not dependent on the thymus for development, and lack the T cell antigen receptor and expression of the CD3 complex on the surface (1,2). Resting or unstimulated NK cells con

12、stitutively express a number of cytokine receptors and may be activated for increased cytolytic activity or cytokine production following stimulation by numerous cytokines (e.g. IL-2, IL-12, IL-15, IL-18, and IL-21) or IFN-/. NK cells are unable to produce any of these cytokinesand are dependent on

13、other cell types to provide these factors (1). The activation of NK cells by these signal factors or cognate interactions with tumor cells also induces NK cells to transcribe and secrete certain chemokines and cytokines including IFN-, GM-CSF, G-CSF, M-CSF, TNF-, and others (3,4). IL-12, a heterodim

14、eric cytokine produced mostly by antigen-presenting cells (APCs) such as stimulated macrophages, monocytes, dendritic cells, neutrophils, and some B cells induces cytokine production, primarily of IFN- by NK and T cells, enhances the cytotoxic activity of NK cells, and promotes differentiation of na

15、ive Th cells into Thl cells (5-7). Thus, IL-12 represents a functional bridge between the early non-specific innate resistance and subsequent antigen-specific adaptive immunity. NK cells have also shown to be activated by IL-12 to kill various tumor cells in a perforin-dependent manner (8). The pore

16、-forming protein perforin is a key effector protein for cytolysis mediated by NK cells and cytotoxic T cells and is preferentially expressed in these cells. Perforin-mediated apoptosis is the principle pathway used by NK cells to kill tumors and virus-infected cells. Although NK cells constitutively

17、 express perforin at a low level, IL-12 and IL-2 have been shown to induce increased accumulation of perforin mRNA in NK cells (9). The biological functions of IL-12 are mediated through specific receptors on NK and T cells. Two IL-12 receptor subunits, IL-12 receptor l (IL-12R 1) and IL-12 receptor

18、 2 (IL-12R 2), have been identified in mouse and human (10-12). IL-12 specifically activates STAT4 in NK and T cells by inducing tyrosine phosphorylation through activation of the IL-12 receptor-associated JAK family kinases, Jak2 and Tyk2 (13). Mushrooms have been valued as flavorful foods and medi

19、cinal substances and are widely sold as nutritional supplements. Polysaccharides from mushrooms have been reported to enhance the immune system. Lentinan from Letinus edodes, schizophyllan (SPG) from Schzophyllum commune, SSG from Sclerotinia sclerotiorum, and PSK from Coriolus versicolor potentiall

20、y exert antitumor effects (14). We previously reported that the (l3)-branched (l6)-glucan, termed the D-Fraction and extracted from the fruiting body of the maitake mushroom (Grifola frondosa), as well as these polysaccharides, is a biological response modifier (BRM), enhanced an immune response thr

21、ough the activation of APCs and T cells (15-17). The administration of D-Fraction to MM-46 tumor-bearing C3H/HeJ mice or colon 26 tumor-bearing BALB/cA mice induced a Th-1-dominant response through IL-12 production by activated APCs (18,19). IL-12 has been shown to possess potent antitumor activity

22、in a wide variety of murine tumor models, and the activity has been demonstrated against tumors of various histologies, including tumors arising from the colon (CT26), kidney (Renca), and lung (3LL); carcinogen-induced sarcoma lines; and melanoma (B16F10) (20-24). In the present study, we investigat

23、ed whether D-Fraction can enhance the activation and cytotoxicity of NK cells in the innate immune responseto the transplantation of MM-46 mammary tumor cells to mice. To use D-Fraction for cancer treatment with IL-12, we investigated the expression of IL-12R l on NK cells by flow cytometry and sugg

24、ested whether D-Fraction-induced IL-12 enhances the cytotoxicity of NK cells by stimulating the IL-12 signaling pathway in NK cells.Materials and methodsAnimals. LPS non-responsive male C3H/HeJ mice (4 weeks old) (CLEA Japan, Inc., Tokyo, Japan) were raised for two weeks before being used for experi

25、ments. Food and water were provided freely to these mice until the experiments.Tumor cells. MM-46 (C3H/He mouse-derived mammary tumor) cells were kindly donated by Dr Kanki Komiyama and maintained in the peritoneal cavity of male C3H/HeJ mice. The murine lymphoma cell line YAC-1 (ATCC number: TIB-16

26、0) was purchased from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan) and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated bovine serum (FBS).Preparation of D-Fraction. D-Fraction was prepared from the dried powder of the fruiting body of maitake mushrooms (Grifola frondosa) (

27、Yukiguni Maitake Co., Ltd., Niigata, Japan), according to the method described in our previous paper (15). The level of LPS contained in D-Fraction was determined using Endospecy ES-20S Set (Seikagaku Corp., Tokyo, Japan), and was 89%.Cytotoxicity assay. Cytotoxicity assays were investigated by measuring and evaluating lactate dehydrogenase (LDH) release from lysed YAC-1 cell (25). Splenic NK, whole spleen, or white blood cells were incubated with YAC-1 cells for 4 h (E:T =25:1 for splenic NK cells, 50:1 for whole spleen cells, and 50:1 for white blood cells). The level of LDH relea