Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, USA.
Am J Physiol Regul Integr Comp Physiol. 2010 Mar;298(3):R584-98. doi: 10.1152/ajpregu.00452.2009. Epub 2009 Dec 9.
Human primary and clonal synovial cells were incubated with glutamate receptor agonists to assess their modulating influence on glutamate receptors N-methyl-d-aspartate (NMDA) NR1 and NR2 and inflammatory cytokines to determine potential for paracrine or autocrine (neurocrine) upregulation of glutamate receptors, as has been shown for bone and chondrocytes. Clonal SW982 synoviocytes constitutively express vimentin, smooth muscle actin (SMA), and NMDA NR1 and NR2. Coincubation (6 h) with glutamate agonists NMDA (5 microM), and the NMDA NR1 glycine site activator (+/-)1-aminocyclopentane-cis-1,3-dicarboxylic acid (5 muM), significantly increases cellular mRNA and protein levels of glutamate receptors, as well as increasing vimentin, SMA, tumor necrosis factor-alpha, and RANTES (regulated on activation, normal T-cell expressed and secreted), assessed qualitatively and quantitatively with nucleotide amplification, image analysis of immunocytochemical staining, fluorescein-activated cell sorting, Western blotting, and immunoassays. Human primary synovial cells harvested from patients with arthritic conditions also constitutively expressed NMDA NR1 with increases after agonist treatment. Glutamate receptor agonist-induced increases were blocked by the noncompetitive glutamate antagonist MK-801 (8 microg/ml) and NR1 blocking antibody. Coincubation with glutamate agonists and phorbol 12-myristate 13-acetate, a protein kinase C activator, significantly enhanced mean levels of TNF-alpha and RANTES in SW982 cell supernatants compared with incubation with either agent alone. Increases were diminished with protein kinase inhibitor and NR1 blocking antibody. The functional activation of glutamate receptors on human synoviocytes establishes a neurogenic cell signaling link between neurotransmitter glutamate released from nerve terminals and target cells in the joint capsule. The influence of glutamate on subsequent release of cellular proinflammatory mediators in non-neural tissue for activation of downstream immune events supports a peripheral neuroimmune link in arthritis.
人原发性和克隆滑膜细胞用谷氨酸受体激动剂孵育，以评估它们对谷氨酸受体 N-甲基-D-天冬氨酸（NMDA）NR1 和 NR2 的调节影响，以及对炎性细胞因子的调节影响，以确定是否存在旁分泌或自分泌（神经内分泌）谷氨酸受体上调的可能性，正如骨和软骨细胞所显示的那样。克隆 SW982 滑膜细胞组成性表达波形蛋白、平滑肌肌动蛋白（SMA）和 NMDA NR1 和 NR2。与谷氨酸激动剂 NMDA（5μM）和 NMDA NR1 甘氨酸位点激动剂（±）1-氨基环戊烷顺-1,3-二羧酸（5μM）共同孵育（6 小时），可显著增加细胞内谷氨酸受体的 mRNA 和蛋白水平，同时增加波形蛋白、SMA、肿瘤坏死因子-α和 RANTES（调节激活，正常 T 细胞表达和分泌），通过核苷酸扩增、免疫细胞化学染色的图像分析、荧光激活细胞分选、Western blot 和免疫测定进行定性和定量评估。从关节炎患者中采集的人原发性滑膜细胞也组成性表达 NMDA NR1，激动剂处理后增加。非竞争性谷氨酸拮抗剂 MK-801（8μg/ml）和 NR1 阻断抗体阻断谷氨酸受体激动剂诱导的增加。与谷氨酸激动剂和佛波醇 12-肉豆蔻酸 13-乙酸（蛋白激酶 C 激活剂）共同孵育，与单独孵育任何一种药物相比，SW982 细胞上清液中 TNF-α和 RANTES 的平均水平显著增强。用蛋白激酶抑制剂和 NR1 阻断抗体处理后，增加减少。谷氨酸受体在人滑膜细胞上的功能激活建立了神经递质谷氨酸从神经末梢释放到关节囊靶细胞之间的神经细胞信号联系。谷氨酸对非神经组织中细胞内前炎性介质随后释放的影响，为关节炎中的外周神经免疫联系提供了支持。
