Kim Joo Han, Studer Rebecca K, Sowa Gwendolyn A, Vo Nam Viet, Kang James D
Department of Orthopaedic Surgery, Ferguson Laboratory for Orthopaedic Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Spine (Phila Pa 1976). 2008 Oct 1;33(21):2253-9. doi: 10.1097/BRS.0b013e318182c35f.
STUDY DESIGN: Anulus fibrosus (AF) cells obtained from patients undergoing surgery were cocultured with macrophage-like cells and production of inflammatory mediators was analyzed by quantitative assay. OBJECTIVE: To investigate the role of macrophages in AF cell production of inflammatory mediators by cytokines stimulation. SUMMARY OF BACKGROUND DATA: Discogenic pain caused by anular disruption is an important cause of low back pain and recent studies show the presence of macrophages in symptomatic discs but not in normal and aging discs. We hypothesize that macrophages play a major role in development of symptomatic disc. METHODS: Human AF cells were cocultured with phorbol myristate acetate stimulated macrophage-like THP-1 cells. The conditioned medium from cells cultured alone or in coculture was assayed for cytokines by Enzyme-linked immunosorbent assay and nitric oxide (NO) by the Greiss method. Using the same outcome measures, comparisons of cell response to cytokines were made among macrophage-like cells, naïve AF cells, and macrophage exposed AF cells. RESULTS.: Tumor necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6, and NO (TNF-alpha: 1.45 +/- 0.29 ng/mL, IL-8: 97.02 +/- 7.94 ng/mL, IL-6: 33.40 +/- 3.55 ng/mL, NO: 8.42 +/- 0.78 micromol/L) were secreted in much greater amounts by cells maintained in coculture compared to macrophages (TNF-alpha: 0.78 +/- 0.12 ng/mL, IL-8: 58.04 +/- 4.44 ng/mL, IL-6: 0.14 +/- 0.03 ng/mL, NO: 0.30 +/- 0.08 micromol/L) or AF cells cultured alone. In addition, IL-6 secretion from AF cells in response to TNF-alpha was up-regulated by coculture, however, IL-6 secretion in response to IL-1 beta was downregulated in a dose-dependent manner. Coculture with macrophages also up-regulated AF cell secretion of IL-8 dose-dependently and downregulated NO to TNF-alpha or IL-1beta stimulation. CONCLUSION: We conclude that exposure to macrophages, as can be expected after anular injury, can result in enhanced response to local inflammation. Although changes were observed in all inflammatory mediators after macrophage exposure, the most significant change was observed in IL-6 and IL-8, implicating these mediators in development of symptomatic disc.
研究设计：将接受手术患者的纤维环（AF）细胞与巨噬细胞样细胞共培养，并通过定量测定分析炎症介质的产生。 目的：通过细胞因子刺激研究巨噬细胞在AF细胞产生炎症介质中的作用。 背景数据总结：纤维环破裂引起的椎间盘源性疼痛是腰痛的重要原因，最近的研究表明，有症状的椎间盘中存在巨噬细胞，而正常和老化的椎间盘中则没有。我们假设巨噬细胞在有症状椎间盘的形成中起主要作用。 方法：将人AF细胞与佛波酯刺激的巨噬细胞样THP-1细胞共培养。通过酶联免疫吸附测定法检测单独培养或共培养细胞的条件培养基中的细胞因子，并通过格里斯方法检测一氧化氮（NO）。使用相同的结果指标，比较巨噬细胞样细胞、未处理的AF细胞和暴露于巨噬细胞的AF细胞对细胞因子的反应。结果：与巨噬细胞（肿瘤坏死因子-α：0.78±0.12 ng/mL，白细胞介素-8：58.04±4.44 ng/mL，白细胞介素-6：0.14±0.03 ng/mL，NO：0.30±0.08 μmol/L）或单独培养的AF细胞相比，共培养的细胞分泌的肿瘤坏死因子-α（TNF-α：1.45±0.29 ng/mL）、白细胞介素-8（IL-8：97.02±7.94 ng/mL）、白细胞介素-6（IL-6：33.40±3.55 ng/mL）和NO（8.42±0.78 μmol/L）的量要多得多。此外，共培养上调了AF细胞对TNF-α反应时IL-6的分泌，然而，AF细胞对IL-1β反应时IL-6的分泌呈剂量依赖性下调。与巨噬细胞共培养还剂量依赖性地上调了AF细胞IL-8的分泌，并下调了AF细胞对TNF-α或IL-1β刺激的NO分泌。 结论：我们得出结论认为，如纤维环损伤后预期的那样，暴露于巨噬细胞可导致对局部炎症的反应增强。尽管巨噬细胞暴露后所有炎症介质均有变化，但IL-6和IL-8的变化最为显著，提示这些介质参与了有症状椎间盘的形成。
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