Chronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored. Fifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1β and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression. Significant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients’ CSF in the presence of LPS. The IL-6 levels showed positive association with pain score (adjusted B estimate = 10.1 (95% Confidence Interval 4.3–15.9); p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase. Higher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of “unknown molecule” in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed.

中文翻译：

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慢性骨关节炎患者的脑脊液诱导人胶质细胞系T98G中白介素6的释放。

就其病理生理机制而言，慢性骨关节炎性疼痛尚未得到很好的理解。活化的神经胶质细胞被认为在维持慢性疼痛中起作用。先前曾观察到T98G胶质母细胞瘤细胞系在患有另一种慢性疼痛症状（带状疱疹后神经痛）的患者中接受脑脊液（CSF）治疗后释放出更高量的白介素6（IL-6）。在这项研究中，我们调查了被诊断患有慢性疼痛的膝骨关节炎患者的脑脊液触发T98G释放促炎性细胞因子，IL-6，IL-1β和肿瘤坏死因子α（TNF-α）的能力。 。还探讨了上游信号的表征。前瞻性招募了15名因慢性膝关节疼痛而进行全膝关节置换的骨关节炎患者和15名没有疼痛的患者进行了脊柱麻醉的其他手术。麻醉期间收集脑脊液。在脂多糖存在下，将CSF加入培养的T98G细胞中。使用酶免疫测定法测量从T98G细胞释放的IL-6，IL-1β和TNF-α。使用CSF触发的T98G细胞裂解物进行抗体阵列和蛋白质印迹，以鉴定可能的信号传导靶标。记录年龄，性别和疼痛评分。使用Mann-Whitney U检验比较两组之间的IL-6释放和蛋白质表达。使用线性回归分析IL-6和疼痛评分之间的关​​联。当存在LPS的情况下，由骨关节炎患者的CSF诱导时，T98G细胞会释放出较高水平的IL-6。IL-6水平与疼痛评分呈正相关（校正后的B估计值= 10.1（95％置信区间4.3-15.9）； p = 0.001）。用骨关节炎疼痛患者CSF诱导的6份合并的T98G细胞裂解物进行的抗体鉴定，鉴定出大于2倍的蛋白质，包括STE20相关激酶衔接蛋白和脾酪氨酸激酶。使用蛋白印迹法对单个CSF触发的T98G细胞裂解物进行的进一步验证显示无明显增加。当存在LPS时，由OA-CSF触发时，T98G的IL-6释放量更高，这表明CSF中存在“未知分子”，这可能对维持骨关节炎人群慢性疼痛的维持至关重要。