Toll样受体在U937细胞的表达及其作用研究
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摘要
目的:急性髓细胞白血病外周血中CD4+CD25high调节性T细胞比例的升高是其免疫功能受损的一个重要机制。TLR8激动剂可有效的阻断调节性T细胞的免疫抑制作用而加强抗肿瘤免疫,提示利用TLR8激动剂阻断调节性T细胞的功能可能是急性髓细胞白血病免疫治疗的一个重要靶点。本实验通过研究人急性髓系白血病细胞U937细胞Toll样受体(TLR1-TLR9)的表达,以及TLR8受体激动剂ssRNA40/LyoVec对其增殖、凋亡、细胞周期和表面分子(CD80、CD86、CD95)的影响,探讨以TLR8为急性髓细胞白血病免疫治疗靶点的可能性。
方法:以人急性髓系白血病细胞U937细胞为研究对象,利用逆转录聚合酶联反应(RT-PCR)法检测其TLR1-TLR9 mRNA的表达,用流式细胞术检测其胞内TLR8的表达。采用CCK-8试剂盒检测了不同浓度的TLR8激动剂ssRNA40/LyoVec(0μg/ml、0.2μg/ml、1μg/ml)在不同的作用时间(24h、48h、72h)处理后U937细胞的增殖活性;1μg/mlssRNA40/LyoVec处理U937细胞72h后,经AnnexinV-FITC/PI双标流式细胞仪检测细胞凋亡,经PI单标流式细胞仪检测细胞周期,流式细胞仪检测其表面分子(CD80、CD86、CD95)的变化。
结果:RT-PCR示U937细胞有TLR1-TLR9的表达,FCM证明了细胞胞内TLR8的表达;CCK-8法示TLR8激动剂ssRNA40/LyoVec对U937细胞的生长有明显的抑制作用,呈时间和浓度依赖性,抑制率可达70%(P<0.05);1μg/ml TLR8激动剂ssRNA40/LyoVec作用于U937细胞72h后,处于G0/G1期细胞比例由(44.67±1.05)%增高到(54.08±1.19)%,但凋亡细胞的比例、表面分子(CD80、CD86、CD95)的表达无明显变化。
结论:TLR1-TLR9可表达在U937细胞,TLR8激动剂ssRNA40/LyoVec具有抑制U937细胞增殖的作用,使细胞阻滞在G0/G1期,但无明显的促凋亡作用,对U937细胞表面分子(CD80、CD86、CD95)的表达无影响。
Objective: Acute myeloid leukemia (AML) is a malignant hematopoietic disorder with considerable impairment of the immune system. We have previously reported that the T-cell-mediated immunity in AML patients could be in part damaged by the increased prevalence of CD4+CD25high regulatory T cells (Tregs). Recent studies demonstrated that synthetic and natural ligands for human TLR8 could completely reverse the suppressive function of Tregs and enhance antitumor immunity, which provides a possible application of TLR8 ligands in immunotherapy for a patient with AML. To explore the possibility, the expression of Toll-like receptors and the effect of TLR8 agonist ssRNA40/LyoVec on the proliferation, apoptosis, cell cycle and expression of CD80, CD86 and CD95 of U937 cells were studied.
Methods: The expression of TLR1-TLR9 in U937 was studied by using reverse transcription polymerase chain reaction (RT-PCR) and the expression of TLR8 was assayed by flow cytometry (FCM). U937 cells were treated with various concentrations ssRNA40/LyoVec (0μg/ml, 0.2μg/ml, 1μg/ml), and the proliferation was determined by CCK-8 kit at different time points (24h, 48h, 72h). To U937 cells treated by 1μg/ml ssRNA40/LyoVec with 72h, apoptosis was analyzed with AnnexinV-FITC/PI staining by FCM, cell cycle was investigated with PI staining by FCM, expression of CD80, CD86 and CD95 was assayed by FCM.
Results: U937 cells expressed TLR1-TLR9. TLR8 agonist ssRNA40/LyoVec could inhibit the growth of U937 cells in time and dose dependent manner and the inhibitory rate could reach 70%(P<0.05). It could also increase the percentage of cells in G0/G1 period. There was no significant difference of percentage of apoptosis cells and the expression of CD80, CD86 and CD95 between control and treated group.
Conclusion: TLRs including TLR1-TLR9 were expressed on U937 cells and TLR8 agonist ssRNA40/LyoVec could inhibit the growth of U937 cells, arrest the cells in G0/G1 period, but have no effect on promoting apoptosis and the expression of CD80, CD86 and CD95.
方法:以人急性髓系白血病细胞U937细胞为研究对象,利用逆转录聚合酶联反应(RT-PCR)法检测其TLR1-TLR9 mRNA的表达,用流式细胞术检测其胞内TLR8的表达。采用CCK-8试剂盒检测了不同浓度的TLR8激动剂ssRNA40/LyoVec(0μg/ml、0.2μg/ml、1μg/ml)在不同的作用时间(24h、48h、72h)处理后U937细胞的增殖活性;1μg/mlssRNA40/LyoVec处理U937细胞72h后,经AnnexinV-FITC/PI双标流式细胞仪检测细胞凋亡,经PI单标流式细胞仪检测细胞周期,流式细胞仪检测其表面分子(CD80、CD86、CD95)的变化。
结果:RT-PCR示U937细胞有TLR1-TLR9的表达,FCM证明了细胞胞内TLR8的表达;CCK-8法示TLR8激动剂ssRNA40/LyoVec对U937细胞的生长有明显的抑制作用,呈时间和浓度依赖性,抑制率可达70%(P<0.05);1μg/ml TLR8激动剂ssRNA40/LyoVec作用于U937细胞72h后,处于G0/G1期细胞比例由(44.67±1.05)%增高到(54.08±1.19)%,但凋亡细胞的比例、表面分子(CD80、CD86、CD95)的表达无明显变化。
结论:TLR1-TLR9可表达在U937细胞,TLR8激动剂ssRNA40/LyoVec具有抑制U937细胞增殖的作用,使细胞阻滞在G0/G1期,但无明显的促凋亡作用,对U937细胞表面分子(CD80、CD86、CD95)的表达无影响。
Objective: Acute myeloid leukemia (AML) is a malignant hematopoietic disorder with considerable impairment of the immune system. We have previously reported that the T-cell-mediated immunity in AML patients could be in part damaged by the increased prevalence of CD4+CD25high regulatory T cells (Tregs). Recent studies demonstrated that synthetic and natural ligands for human TLR8 could completely reverse the suppressive function of Tregs and enhance antitumor immunity, which provides a possible application of TLR8 ligands in immunotherapy for a patient with AML. To explore the possibility, the expression of Toll-like receptors and the effect of TLR8 agonist ssRNA40/LyoVec on the proliferation, apoptosis, cell cycle and expression of CD80, CD86 and CD95 of U937 cells were studied.
Methods: The expression of TLR1-TLR9 in U937 was studied by using reverse transcription polymerase chain reaction (RT-PCR) and the expression of TLR8 was assayed by flow cytometry (FCM). U937 cells were treated with various concentrations ssRNA40/LyoVec (0μg/ml, 0.2μg/ml, 1μg/ml), and the proliferation was determined by CCK-8 kit at different time points (24h, 48h, 72h). To U937 cells treated by 1μg/ml ssRNA40/LyoVec with 72h, apoptosis was analyzed with AnnexinV-FITC/PI staining by FCM, cell cycle was investigated with PI staining by FCM, expression of CD80, CD86 and CD95 was assayed by FCM.
Results: U937 cells expressed TLR1-TLR9. TLR8 agonist ssRNA40/LyoVec could inhibit the growth of U937 cells in time and dose dependent manner and the inhibitory rate could reach 70%(P<0.05). It could also increase the percentage of cells in G0/G1 period. There was no significant difference of percentage of apoptosis cells and the expression of CD80, CD86 and CD95 between control and treated group.
Conclusion: TLRs including TLR1-TLR9 were expressed on U937 cells and TLR8 agonist ssRNA40/LyoVec could inhibit the growth of U937 cells, arrest the cells in G0/G1 period, but have no effect on promoting apoptosis and the expression of CD80, CD86 and CD95.
引文
1. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol, 2003, 21:335-376
2. Huang B, Zhao J, Li H,et al. Toll-like receptors on tumor cells facilitate evasion of immune surveillance. Cancer Res, 2005, 65: 5009 - 5014
3. Salaun B, Coste I, Rissoan MC, et al. TLR3 can directly trigger apoptosis in human cancer cells. J Immunol, 2006, 176: 4894 - 4901
4. Bohnhorst J, Rasmussen T, Moen SH, et al. Toll-like receptors mediate proliferation and survival of multiple myeloma cells. Leukemia, 2006, 20: 1138 - 1144
5. Wang X, Zheng J, Liu J, et al. Increased population of CD4(+)CD25(high) regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients. Eur J Haematol, 2005, 75: 468 - 476
6. Peng G, Guo Z, Kiniwa Y, et al. Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science, 2005, 309: 1380 - 1384
7. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004, 303: 1526 - 1529
8. Beyer M, Schultze JL. Regulatory T cells in cancer. Blood, 2006, 108: 804 - 811.
9. Wang RF. Regulatory T Cells and Toll-Like Receptors in Cancer Therapy. Cancer Res, 2006, 66: 4987 - 4990.
10. Tomic J, White D, Shi Y,et al. Sensitization of IL-2 signaling through TLR-7 enhances B lymphoma cell immunogenicity. J Immunol, 2006, 176: 3830 - 3839
11. Corthals SL, Wynne K, She K, et al. Differential immune effects mediated by Toll-like receptors stimulation in precursor B-cell acute lymphoblastic leukaemia. Br J Haematol, 2006, 132: 452 - 458.
12. Spaner DE, Miller RL, Mena J, et al. Regression of lymphomatous skin deposits in a chronic lymphocytic leukemia patient treated with the Toll-like receptor-7/8 agonist, imiquimod. Leuk Lymphoma, 2005, 46: 935 - 939
13. Spaner DE, Masellis A. Toll-like receptor agonists in the treatment of chronic lymphocytic leukemia. Leukemia, 2007, 21: 53 - 60
14. Ma Y, Li J, Chiu I, et al. Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. J Cell Biol, 2006, 175: 209 -215
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3. Salaun B, Coste I, Rissoan MC, et al. TLR3 can directly trigger apoptosis in human cancer cells. J Immunol, 2006, 176: 4894 - 4901
4. Bohnhorst J, Rasmussen T, Moen SH, et al. Toll-like receptors mediate proliferation and survival of multiple myeloma cells. Leukemia, 2006, 20: 1138 - 1144
5. Wang X, Zheng J, Liu J, et al. Increased population of CD4(+)CD25(high) regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients. Eur J Haematol, 2005, 75: 468 - 476
6. Peng G, Guo Z, Kiniwa Y, et al. Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science, 2005, 309: 1380 - 1384
7. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004, 303: 1526 - 1529
8. Beyer M, Schultze JL. Regulatory T cells in cancer. Blood, 2006, 108: 804 - 811.
9. Wang RF. Regulatory T Cells and Toll-Like Receptors in Cancer Therapy. Cancer Res, 2006, 66: 4987 - 4990.
10. Tomic J, White D, Shi Y,et al. Sensitization of IL-2 signaling through TLR-7 enhances B lymphoma cell immunogenicity. J Immunol, 2006, 176: 3830 - 3839
11. Corthals SL, Wynne K, She K, et al. Differential immune effects mediated by Toll-like receptors stimulation in precursor B-cell acute lymphoblastic leukaemia. Br J Haematol, 2006, 132: 452 - 458.
12. Spaner DE, Miller RL, Mena J, et al. Regression of lymphomatous skin deposits in a chronic lymphocytic leukemia patient treated with the Toll-like receptor-7/8 agonist, imiquimod. Leuk Lymphoma, 2005, 46: 935 - 939
13. Spaner DE, Masellis A. Toll-like receptor agonists in the treatment of chronic lymphocytic leukemia. Leukemia, 2007, 21: 53 - 60
14. Ma Y, Li J, Chiu I, et al. Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. J Cell Biol, 2006, 175: 209 -215
1. O’Neill LA. How Toll-like receptors signal: what we know and what we don’t know. Curr Opin Immunol, 2006, 25: 687 - 693
2. McGettrick AF, O’Neill LA. The expanding family of MyD88-like adapters in TLR signals transduction. Mol Immunol, 2004, 41: 577 - 582
3. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett, 2003, 85: 85 - 95
4. Beg AA. Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol, 2002, 23: 509 - 512
5. Doyle SE, O’connell R, Vaid Y, et al. Toll-like receptor 3 mediates a more potent antiviral response than toll-like receptor 4. J Immunol, 2003, 170: 3565 - 3571
6. Hwang D. Modulation of the expression of cyclooxygenase-2 by fatty acids mediated through Toll-like receptor 4-derived signaling pathways. FASEB J, 2001, 15: 2556 - 2564
7. Abel B, Thieblemont N, Quensniaux VT, et al. Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice. J Immunol, 2002, 169: 3155 - 3162
8. Ishii K, Takamura N, Shimohara E, et al. Intracellular cytokine analysis of CD4-positive T cells predictive of sustained response to interferon therapy for patients with chronic hepatitis C. Dig Dis Sci, 2002, 47: 778 - 783
9. Lorenz E, Mira JP, Frees KL, et al. Relevance of mutations in the TLR4 receptor in patients with gram-negative septic hock. Arch Intern Med, 2002, 162: 1028 - 1032
10. Kobayashi N, Takesue M, Kobayashi M, et al. Evaluation of toll-like receptor 4 gene expression of immortalized human liver cell lines. Transplant Proc, 2003, 35: 431 - 432
11. Kadowaki N, Liu YJ. Natural type I interferon-producing cells as a link between innate and adaptive immunity. Hum Immunol, 2002, 63:1126 - 1132
12. Calkins CM, Barsness K, Bensard DD, et al. Toll-like receptor 4 signaling mediates pulmonary neutrophil sequestration in response to gram-positive bacterial enterotoxin. J Surg Res, 2002, 104: 124 - 130
13. Liauet L, Deb A, Pacher P. The flagellin-TLR5 axis: therapeutic opportunities. Drug News Perspect, 2002, 15: 397– 409
14. Heil F, Hemmi H, Hochrein H,et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004, 303: 1526 - 1529.
15. HemmiH, Kaisho T, Takeuchi O, et al. Small anti-viral compounds active immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol, 2002, 3: 190 - 200
16. O’neill LA. TLR7 and antiviral immunity. Trends Immunol, 2002, 23: 234 - 239
17. O’Neill L A, Dunne A, Edjeback M,et al.Mal and MyD88: adapter proteins involved in signal transduction by toll-like receptors. J Endotoxin Res, 2003, 9: 55 - 59.
18. Kaisho T, Akira S. Dendritic-cell function in toll-like receptor and MyD88-knockout mice. Trends Immunol, 2001, 22 : 78 - 83
19. Furusako S, Takahashi S, Mori S, et al. Protection of mice from LPS-induced shock by CD14 antisense oligonucleotide . Acta Med, 2001, 55: 105 - 115
20. Zuanya C, Hastewell J, Walker C. Toll-like receptors as potential therapeutic targets for multiple diseases. Nat Rev Drug Discov, 2002, 1: 797 - 807
21. Szabo C. Role of flagellin in the pathogenesis of shock and acute respiratory distress syndrome: therapeutic opportunities. Crit Care Med, 2003, 31: S39 - S45.
22. Poland GA, Murray D, Bonilla-Guerrero R. New vaccine development. BMJ, 2002, 324: 1315 - 1319
23. SchmittC, Humeny A, Becker CM, et al. Polymorphisms of TLR4: rapid genotyping and reduced response to lipopolysaccharide of TLR4 mutant alleles. Clin Chem, 2002, 48: 1661 - 1667
24. Mak TW, Yehw WC.Immunology: a block at the toll gate. Nature, 2002, 418: 835 - 836.
25. Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol, 2004, 5: 975 - 995
26. Child NJ, Yang IA, Pulletz MC, et al. Polymorphisms in Toll-like receptor4 and the systemic inflammatory response syndrome. Biochem Soc Trans, 2003, 31: 652 - 653
27. Smirnova I, Mann N, Dols A, et al. Assay of locus-specific genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility. Proc Natl Acad Sci USA, 2003, 100: 6075 - 608
28. Ogus AC, Yoldas B, Ozdemir T, et al. The Arg753GIn polymorphism of the human Toll-like receptor 2 gene in tuberculosis disease. Eur Respir J, 2004, 23: 219 - 223
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