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重金属是一类典型的环境污染物,广泛存在于土壤、淡水、空气等环境介质中,通过食物、饮水和吸烟等途径暴露后可在肝、脑、肾、心脏等重要器官中富集并长久滞留[1]. 目前,有关重金属毒性效应的研究已成为环境毒理学的重要组成部分,包括血液毒性、心血管疾病、神经毒性、肾毒性、癌症等[2]. 其中,重金属与免疫系统的相互作用可对人体健康产生不利影响,导致自身免疫性疾病和各种癌症的风险增加[3]. 有研究表明,重金属砷(As)、镉(Cd)和铅(Pb)可以上调特定炎症介质和标志物的表达,从而改变淋巴细胞功能,促进细胞因子和免疫球蛋白的产生[3]. 树突状细胞(Dendritic cell, DC)作为主要的抗原呈递细胞之一,在肿瘤微环境中发挥着免疫调控作用. 已有研究发现,As可以通过下调骨髓源性树突状细胞(Bone marrow-derived dendritic cell, BMDC)活化分子、促炎因子和T淋巴细胞辅助因子的表达进而引起免疫耐受反应[4]. Cd通过下调活化分子MHC II和CD40的表达,抑制IL-12p70的分泌进而抑制BMDC细胞的成熟[5]. Pb则可抑制BMDC细胞的发育,但同时又促进细胞分泌大量促炎因子,从而引起强烈的促炎反应[6 − 7]. 重金属是否影响DC细胞的吞噬、改变其抗原结合功能,尚不清楚;同时,不同重金属之间对DC细胞的毒性和功能影响存在怎样的差异,也亟需探讨.
本研究通过Alamar blue法测定BMDC细胞的活性,采用活性氧(Reactive oxygen species, ROS)和丙二醛检测试剂盒检测细胞膜的通透性,并利用流式细胞术检测BMDC细胞的凋亡、活化、吞噬以及抗原结合能力,最后通过荧光定量PCR检测BMDC细胞促炎/抗炎细胞因子的胞内表达水平. 结果表明,不同重金属对树突状细胞的功能改变存在差异.
重金属砷、镉和铅对树突状细胞的差异化功能损伤
Diverse effects of heavy metals on the functions of dendritic cells
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摘要: 为了探讨重金属砷、镉和铅对树突状细胞的毒性作用和功能影响,以骨髓源性树突状细胞(Bone marrow-derived dendritic cell,BMDC)为研究对象,采用Alamar blue法测定BMDC细胞的活性,采用流式细胞术检测BMDC细胞的凋亡、活化、吞噬以及抗原结合能力,并对细胞活性氧和丙二醛进行测定;采用荧光定量PCR检测BMDC细胞促炎/抗炎细胞因子的胞内表达水平. 结果发现,3种重金属均可引起BMDC细胞活性降低(其中镉和铅可促进细胞凋亡)、导致细胞膜通透性改变并抑制细胞的活化;但3种重金属对BMDC细胞的功能影响存在差异,体现在砷可抑制BMDC细胞的吞噬功能,而铅和高浓度的镉则抑制BMDC细胞的抗原结合能力,同时促进促炎细胞因子的表达. 上述结果表明,不同重金属对树突状细胞具有相似的细胞毒性,但对其功能的影响存在差异,其中,砷主要影响未成熟树突状细胞的吞噬功能,铅和镉则主要影响成熟树突状细胞的抗原结合功能.Abstract: In order to investigate the toxic effects and functional inhibition of typical heavy metals on dendritic cells, bone marrow-derived dendritic cells (BMDCs) were treated with arsenic (As), cadmium (Cd), or lead (Pb). BMDCs were stained with Alamar Blue to examine cell viability. Flow cytometry was employed to investigate the apoptosis, cell activation, phagocytosis, antigen binding capacity, and the levels of cellular reactive oxygen species and malondialdehyde for BMDCs. By using RT-qPCR, the expression levels of pro- and anti-inflammatory cytokines in BMDCs were also determined. The results demonstrated that all three heavy metals reduced BMDC activity, in which Cd and Pb promoted the apoptosis of BMDCs. Exposure to As, Cd or Pb altered cell membrane permeability and prevented the activation of BMDCs. However, As, Cd and Pb showed varied effects on BMDC functions, in which As prevented the phagocytosis capability while Pb and high-dosed Cd prevented the antigen-binding capacity of BMDCs. In conclusion, As, Cd and Pb exhibited comparable cytotoxic effects on dendritic cells, but varied impacts on the functions of dendritic cells.
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Key words:
- heavy metals /
- dendritic cells /
- cell activation /
- antigen recognition /
- phagocytosis
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表 1 不同细胞因子的定量PCR引物序列
Table 1. Primers of qPCR for testing the expression of 12 cytokines
名称 5′端引物序列(5′ to 3′) 3′端引物序列(5′ to 3′) CCR7 TGTACGAGTCGGTGTGCTTC GGTAGGTATCCGTCATGGTCTTG IFN-γ ATGAACGCTACACACTGCATC CCATCCTTTTGCCAGTTCCTC TNF-α CCTGTAGCCCACGTCGTAG GGGAGTAGACAAGGTACAACCC IL-1α TTGGTTAAATGACCTGCAACA GAGCGCTCACGAACAGTTG IRF7 GAGACTGGCTATTGGGGGAG GACCGAAATGCTTCCAGGG IL-6 TCTAATTCATATCTTCAACCAAGAGG TGGTCCTTAGCCACTCCTTC IL-12 CTGTGCCTTGGTAGCATCTATG GCAGAGTCTCGCCATTATGATTC IL-10 GCTGGACAACATACTGCTAACC ATTTCCGATAAGGCTTGGCAA IL-4 CCCCAGCTAGTTGTCATCCTG CAAGTGATTTTTGTCGCATCCG NF-κB-P50 GGAGGCATGTTCGGTAGTGG CCCTGCGTTGGATTTCGTG NF-κB-P65 AGGCTTCTGGGCCTTATGTG TGCTTCTCTCGCCAGGAATAC β-actin GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT -
[1] FU Z S, XI S H. The effects of heavy metals on human metabolism[J]. Toxicology Mechanisms and Methods, 2020, 30(3): 167-176. doi: 10.1080/15376516.2019.1701594 [2] JAISHANKAR M, TSETEN T, ANBALAGAN N, et al. Toxicity, mechanism and health effects of some heavy metals[J]. Interdisciplinary Toxicology, 2014, 7(2): 60-72. doi: 10.2478/intox-2014-0009 [3] EBRAHIMI M, KHALILI N, RAZI S, et al. Effects of lead and cadmium on the immune system and cancer progression[J]. Journal of Environmental Health Science & Engineering, 2020, 18(1): 335-343. [4] LI J L, GUO Y Y, DUAN X X, et al. Heme oxygenase-1 (HO-1) assists inorganic arsenic-induced immune tolerance in murine dendritic cells[J]. Chemosphere, 2021, 264: 128452. doi: 10.1016/j.chemosphere.2020.128452 [5] CHAKRABORTY K, CHATTERJEE S, BHATTACHARYYA A. Modulation of phenotypic and functional maturation of murine bone-marrow-derived dendritic cells (BMDCs) induced by cadmium chloride[J]. International Immunopharmacology, 2014, 20(1): 131-140. doi: 10.1016/j.intimp.2014.02.015 [6] GAO D H, MONDAL T K, LAWRENCE D A. Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses[J]. Toxicology and Applied Pharmacology, 2007, 222(1): 69-79. doi: 10.1016/j.taap.2007.04.001 [7] METRYKA E, CHIBOWSKA K, GUTOWSKA I, et al. Lead (Pb) exposure enhances expression of factors associated with inflammation[J]. International Journal of Molecular Sciences, 2018, 19(6): 1813. doi: 10.3390/ijms19061813 [8] MA J, HE P, ZHAO C F, et al. A designed α-GalCer analog promotes considerable Th1 cytokine response by activating the CD1d-iNKT axis and CD11b-positive monocytes/macrophages[J]. Advanced Science, 2020, 7(14): 2000609. doi: 10.1002/advs.202000609 [9] MACOCH M, MORZADEC C, FARDEL O, et al. Inorganic arsenic impairs differentiation and functions of human dendritic cells[J]. Toxicology and Applied Pharmacology, 2013, 266(2): 204-213. doi: 10.1016/j.taap.2012.11.008 [10] METRYKA E, KUPNICKA P, KAPCZUK P, et al. Lead (Pb) accumulation in human THP-1 monocytes/macrophages in vitro and the influence on cell apoptosis[J]. Biological Trace Element Research, 2021, 199(3): 955-967. doi: 10.1007/s12011-020-02215-7 [11] CHEN J F, JIN Z Q, ZHANG S Q, et al. Arsenic trioxide elicits prophylactic and therapeutic immune responses against solid tumors by inducing necroptosis and ferroptosis[J]. Cellular & Molecular Immunology, 2023, 20(1): 51-64. [12] GOTTSCHALK C, METTKE E, KURTS C. The role of invariant natural killer T cells in dendritic cell licensing, cross-priming, and memory CD8(+) T cell generation[J]. Frontiers in Immunology, 2015, 6: 379. [13] MERAD M, SATHE P, HELFT J, et al. The dendritic cell lineage: Ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting[J]. Annual Review of Immunology, 2013, 31: 563-604. doi: 10.1146/annurev-immunol-020711-074950 [14] REYNISSON B, ALVAREZ B, PAUL S, et al. NetMHCpan-4.1 and NetMHCIIpan-4.0: Improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data[J]. Nucleic Acids Research, 2020, 48(W1): W449-W454. doi: 10.1093/nar/gkaa379 [15] RADHAKRISHNAN S, CELIS E, PEASE L R. B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(32): 11438-11443. [16] TURNBULL E, MacPHERSON G. Immunobiology of dendritic cells in the rat[J]. Immunological Reviews, 2001, 184(1): 58-68. doi: 10.1034/j.1600-065x.2001.1840106.x [17] MORRIS G, GEVEZOVA M, SARAFIAN V, et al. Redox regulation of the immune response[J]. Cellular & Molecular Immunology, 2022, 19(10): 1079-1101. [18] PLANTINGA M, GUILLIAMS M, VANHEERSWYNGHELS M, et al. Conventional and monocyte-derived CD11b+ dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen[J]. Immunity, 2013, 38(2): 322-335. doi: 10.1016/j.immuni.2012.10.016 [19] LEPLINA O Y, TYRINOVA T V, TIKHONOVA M A, et al. Interferon alpha induces generation of semi-mature dendritic cells with high pro-inflammatory and cytotoxic potential[J]. Cytokine, 2015, 71(1): 1-7. doi: 10.1016/j.cyto.2014.07.258 [20] ARNOLD I C, MATHISEN S, SCHULTHESS J, et al. CD11c+ monocyte/macrophages promote chronic Helicobacter hepaticus-induced intestinal inflammation through the production of IL-23[J]. Mucosal Immunology, 2016, 9(2): 352-363. doi: 10.1038/mi.2015.65 [21] BANCHEREAU J, PASCUAL V, O'GARRA A. From IL-2 to IL-37: The expanding spectrum of anti-inflammatory cytokines[J]. Nature Immunology, 2012, 13(10): 925-931. doi: 10.1038/ni.2406 [22] AMON L, LEHMANN C H K, BARANSKA A, et al. Transcriptional control of dendritic cell development and functions[J]. International Review of Cell and Molecular Biology, 2019, 349: 55-151.