Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison Street, Suite 300, Chicago, IL 60612, USA; Department of Orthopedic and Traumatology, Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Rd, Beitou District, Taipei City, Taiwan 11217, ROC; School of Medicine, National Yang-Ming University, No.155, Sec. 2, Linong St, Beitou District, Taipei City, Taiwan 112, ROC.
Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison Street, Suite 300, Chicago, IL 60612, USA.
Spine J. 2020 Dec;20(12):2025-2036. doi: 10.1016/j.spinee.2020.07.001. Epub 2020 Jul 13.
Targeting chemokines or chemokine receptors is a promising treatment strategy for diseases with chronic inflammation such as rheumatoid arthritis and discogenic pain. Identifying specific molecules and determining their effectiveness in animal models are the first steps in developing these treatments. Macrophage markers have been detected in the intervertebral disc tissues of patients with disc degenerative disease and discogenic pain and in different animal models. Macrophage recruitment into the disc may play a role in initiation of inflammation and if unresolved may lead to chronic inflammation and subsequent back pain.
The objectives of these studies are to (1) identify chemokine receptor antagonists that can block macrophage migration induced by disc cells in vitro and (2) determine if intradiscal treatment with these antagonists can reduce disc inflammation and degeneration in vivo.
In vitro migration assays were used to test effectiveness of chemokine receptor antagonists to block macrophage migration induced by disc cells. The rabbit annular puncture model was used to test for anti-inflammatory and regenerative effects of chemokine receptor antagonist treatment in vivo.
In vitro - THP-1 human monocytic cell line and freshly isolated rabbit primary splenocytes were assayed for migration using 3 µm Corning Transwell inserts with conditioned media of interleukin (IL)-1β treated human or rabbit disc cells. Inhibition of macrophage migration was evaluated using different concentrations of small molecule antagonists of C-C chemokine receptor (CCR)1 and CCR2. In vivo - New Zealand White rabbits (n=40) underwent disc puncture and intradiscal treatment with saline, CCR1 or CCR2 antagonists within the same procedure. X-ray and magnetic resonance (MR) images and serum samples were taken for disc height, MRI grade and IL-8 serum level analyses. Intervertebral discs were isolated for RNA analysis of inflammatory and disc phenotypic markers and for immunohistochemical analysis of macrophage marker, RAM11. The outcome measures were compared between the three treatment groups. These studies were funded by a research grant from AO Foundation, Switzerland (Project no S-14-86A; 120000 CHF). CCR1 and CCR2 antagonists were kindly provided by ChemoCentryx (Mountain View, CA).
In vitro migration assays showed that THP-1 migration induced by disc cells was blocked by CCR2 antagonist more effectively than CCR1 antagonist, while rabbit splenocyte migration was inhibited by CCR1 antagonist and not the other. In the rabbit annular puncture model, rabbit discs treated with CCR1 antagonist had significantly better MRI grades than those treated with CCR2 antagonist at 6 weeks post-treatment. Gene expression studies demonstrate that discs treated with CCR1 or CCR2 antagonists expressed less inflammatory markers than saline-treated discs at 3 weeks post-treatment. Although CCR2 antagonist treatment did not reduce inflammatory marker expression at 6 weeks, discs treated with CCR1 antagonist expressed less inflammatory markers and also a higher ratio of collagen type 2 to collagen type 1 genes indicating favorable disc matrix production. There were no significant differences between all three treatment groups in regards to disc height indexes, IL-8 serum levels or macrophage marker detection.
These studies have identified that small molecule antagonists against CCR2 and CCR1 were respectively effective in blocking THP-1 and rabbit splenocyte migration induced by disc cells in vitro. Further, both CCR2 and CCR1 antagonist intradiscal treatments were effective in reducing disc inflammation at an early time point of 3 weeks. Lastly, only CCR1 antagonist demonstrated anti-inflammatory effects and better MRI grades at 6 weeks.
Our preclinical studies demonstrate that CCR1 and CCR2 antagonist delivery through intradiscal injection is sufficient to reduce disc inflammation at early time points, whereas CCR1 antagonists had longer term anti-inflammatory effects. Clinical studies have found that CCR1 antagonist was safe, tolerable and clinically active in reducing inflammation in rheumatoid arthritis patients. These studies suggest that CCR1 antagonist may be a promising biological treatment to reduce disc inflammation that translates to back pain relief.
靶向趋化因子或趋化因子受体是治疗类风湿关节炎和椎间盘源性疼痛等慢性炎症性疾病的一种有前途的治疗策略。确定特定的分子并在动物模型中确定其有效性是开发这些治疗方法的第一步。在椎间盘退行性疾病和椎间盘源性疼痛患者的椎间盘组织以及不同的动物模型中，已经检测到巨噬细胞标志物。巨噬细胞募集到椎间盘可能在炎症的启动中起作用，如果未解决，可能导致慢性炎症和随后的背痛。
这些研究的目的是：（1）鉴定可阻断体外椎间盘细胞诱导的巨噬细胞迁移的趋化因子受体拮抗剂；（2）确定这些拮抗剂在体内椎间盘内治疗是否可以减少椎间盘炎症和退变。
体外迁移试验用于测试趋化因子受体拮抗剂阻断椎间盘细胞诱导的巨噬细胞迁移的有效性。采用兔环状穿刺模型，研究趋化因子受体拮抗剂治疗对体内椎间盘炎症和再生的作用。
体外：用 3 µm Corning Transwell 插入物检测经白细胞介素（IL）-1β处理的人或兔椎间盘细胞条件培养基诱导的 THP-1 人单核细胞系和新鲜分离的兔原代脾细胞的迁移。使用不同浓度的 C-C 趋化因子受体（CCR）1 和 CCR2 小分子拮抗剂评估巨噬细胞迁移的抑制情况。体内：新西兰白兔（n=40）行椎间盘穿刺，在同一手术过程中用生理盐水、CCR1 或 CCR2 拮抗剂进行椎间盘内治疗。进行 X 射线和磁共振（MR）成像及血清样本采集，以分析椎间盘高度、MRI 分级和 IL-8 血清水平。分离椎间盘中的 RNA，分析炎症和椎间盘表型标志物，并进行巨噬细胞标志物 RAM11 的免疫组织化学分析。比较三组治疗的结果。这些研究由瑞士 AO 基金会的研究基金（项目编号 S-14-86A；120000 CHF）资助。CCR1 和 CCR2 拮抗剂由 ChemoCentryx（加利福尼亚州山景城）提供。
体外迁移试验表明，CCR2 拮抗剂比 CCR1 拮抗剂更有效地阻断椎间盘细胞诱导的 THP-1 迁移，而兔脾细胞迁移则被 CCR1 拮抗剂抑制，而非其他拮抗剂。在兔环状穿刺模型中，在治疗后 6 周时，用 CCR1 拮抗剂治疗的兔椎间盘的 MRI 分级明显优于用 CCR2 拮抗剂治疗的椎间盘。基因表达研究表明，在治疗后 3 周时，用 CCR1 或 CCR2 拮抗剂治疗的椎间盘表达的炎症标志物少于生理盐水治疗的椎间盘。尽管 CCR2 拮抗剂治疗在 6 周时没有降低炎症标志物的表达，但用 CCR1 拮抗剂治疗的椎间盘表达的炎症标志物较少，并且胶原类型 2 与胶原类型 1 基因的比例也较高，表明有利的椎间盘基质生成。在椎间盘高度指数、IL-8 血清水平或巨噬细胞标志物检测方面，三组治疗之间没有显著差异。
这些研究已经确定，体外分别针对 CCR2 和 CCR1 的小分子拮抗剂有效阻断了椎间盘细胞诱导的 THP-1 和兔脾细胞的迁移。此外，CCR2 和 CCR1 拮抗剂的椎间盘内治疗在 3 周的早期时间点都能有效减轻椎间盘炎症。最后，只有 CCR1 拮抗剂在 6 周时表现出抗炎作用和更好的 MRI 分级。
我们的临床前研究表明，通过椎间盘内注射给予 CCR1 和 CCR2 拮抗剂足以在早期减轻椎间盘炎症，而 CCR1 拮抗剂具有更长的抗炎作用。临床研究发现，CCR1 拮抗剂在减轻类风湿关节炎患者炎症方面是安全、耐受和有效的。这些研究表明，CCR1 拮抗剂可能是一种有前途的生物治疗方法，可减轻椎间盘炎症，从而缓解背痛。
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