Role of CD14-positive cells in inflammatory cytokine and pain-related molecule expression in human degenerated intervertebral discs
Role of CD14-positive cells in inflammatory cytokine and pain-related molecule expression in human degenerated intervertebral discs
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DOI：
10.1002/jor.24839
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发表时间：
2020-09-23
影响因子：
2.8
通讯作者：
Takaso, Masashi
中科院分区：
文献类型：
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作者：
Miyagi, Masayuki;Uchida, Kentaro;Takaso, Masashi
多项人类和动物研究表明，退行性或损伤的椎间盘（IVD）中炎性细胞因子和其他疼痛相关分子的上调可能导致椎间盘源性腰痛（LBP）。我们以前报道过，在小鼠受损的IVD中，巨噬细胞产生炎性细胞因子，但不产生其他疼痛相关分子。CD 14是主要由巨噬细胞表达的单核细胞标志物。本研究的目的是评估CD 14阳性细胞在人类退行性IVD中炎性细胞因子和疼痛相关分子表达中的作用。从14名患者中采集IVD样本，包括10名腰椎管狭窄症患者、4名成人脊柱畸形患者和1名脊柱椎间融合术期间腰椎间盘突出症患者。获得收获的IVD来源的单核细胞，并使用CD 14抗体和链霉亲和素标记的磁珠分离CD 14阳性（+）和CD 14阴性（-）细胞。采用定量聚合酶链反应（qPCR）测定CD 14（+）和CD 14（-）细胞中炎性细胞因子信使RNA（mRNA），包括肿瘤坏死因子x251（TNFA）、白细胞介素-1 β（IL 1B）和IL 6，并比较它们的表达水平。为了评价控制疼痛相关分子mRNA表达调节的因素，用重组人TNF-x251刺激培养的来自IVD的CD 14（-）和CD 14（+）细胞;使用qPCR测定IL-1 β和疼痛相关分子（包括降钙素基因相关肽（CGRP）和神经生长因子（NGF））的水平。与CD 14（-）细胞相比，CD 14（+）细胞中的TNFA、IL 1B、IL 6和NGF水平显著升高（TNFA，p = 0.006; IL 1B，p = 0.017; IL 6，p = 0.010;NGF，p = 0.027）。TNF α刺激后，CD 14（-）和CD 14（+）细胞中的NGF水平显著增加（CD 14（-），p = 0.003; CD 14（+），p <0.001），CD 14（-）IVD细胞中的CGRP显著增加（p = 0.040）。IL 1B刺激后，CD 14（-）细胞中的NGF水平显著升高（p = 0.004）。在人退变IVD中，CD 14（+）细胞比CD 14（-）细胞表达TNF A、IL 1B、IL 6和NGF。另外，TNF α刺激促进了CD 14（-）细胞中NGF和CGRP的表达上调。这些结果表明，CD 14（+）细胞直接或间接参与了人类退行性IVD中炎性细胞因子和疼痛相关分子的表达。CD 14（+）细胞可能在人类慢性椎间盘源性LBP的病理机制中起重要作用。
Multiple human and animal studies suggest that the upregulation of inflammatory cytokines and other pain-related molecules in degenerated or injured intervertebral discs (IVDs) may cause discogenic low back pain (LBP). We previously reported that macrophages in injured IVD in mice produced inflammatory cytokines, but not other pain-related molecules. CD14 is a monocyte marker expressed mainly by macrophages. The aim of the current study was to evaluate the role of CD14-positive cells in inflammatory cytokine and pain-related molecule expression in human degenerated IVD. IVD samples were harvested from 14 patients, including 10 with lumbar spinal stenosis, four with adult spinal deformity, and one with lumbar disc herniation during spinal interbody fusion surgery. Harvested IVD-derived mononuclear cells were obtained and CD14-positive (+) and CD14-negative (-) cells were separated using CD14 antibody and streptavidin-labeled magnetic beads. Inflammatory cytokines messenger RNA (mRNA) in the CD14(+) and CD14(-) cells, including tumor necrosis factor x251; (TNFA), in, terleukin-1 beta (IL1B) andIL6, were determined using quantitative polymerase chain reaction (qPCR) and their expression levels were compared. To evaluate factors controlling the regulation of pain-related molecules mRNA expression, cultured CD14(-) and CD14(+) cells from IVDs were stimulated with recombinant human TNF-x251; and IL-1 beta and levels of pain-related molecules, including calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) were determined using qPCR. Levels ofTNFA, IL1B, IL6, andNGFin CD14(+) cells were significantly increased compared with those in CD14(-) cells (TNFA, p = 0.006;IL1B, p = .017;IL6, p = .010;NGF, p = .027). FollowingTNFAstimulation,NGFlevels were significantly increased in CD14(-) and CD14(+) cells (CD14(-),p = .003; CD14(+),p < .001) and CGRP was significantly increased in CD14(-) IVD cells (p = .040). FollowingIL1Bstimulation,NGFlevels were significantly increased in CD14(-) cells (p = .004). CD14(+) cells had higherTNFA, IL1B, IL6, andNGFexpressions than CD14(-) cells in human degenerated IVDs. Additionally,TNFAstimulation promoted the upregulation ofNGFandCGRPin CD14(-) cells. These findings suggested that CD14(+) cells directly and indirectly contributed to inflammatory cytokine and pain-related molecule expression in human degenerated IVD. CD14(+) cells might be important in the pathological mechanism of chronic discogenic LBP in humans.