Elisia Ingrid, Pae Han Bee, Lam Vivian, Cederberg Rachel, Hofs Elyse, Krystal Gerald
The Terry Fox Laboratory, British Columbia Cancer Agency, Canada.
The Terry Fox Laboratory, British Columbia Cancer Agency, Canada.
J Immunol Methods. 2018 Jan;452:26-31. doi: 10.1016/j.jim.2017.10.004. Epub 2017 Oct 16.
The RAW264.7 mouse macrophage cell line is used extensively to carry out in vitro screens for immunomodulators. Compounds that are effective at reducing the expression of pro-inflammatory cytokines or nitric oxide (NO) from lipopolysaccharide (LPS)-stimulated RAW264.7 cells are often considered candidate anti-inflammatory agents for humans. There is, however, very little data on the reliability of this screen to identify bona fide human immunomodulators. We compared the efficacy of 37 purported immunomodulators to modulate LPS or E. coli-induced inflammatory responses in RAW264.7 cell, whole human blood and human peripheral blood mononuclear cell (PBMC) assays. Interestingly, there was no significant correlation (R=0.315) between the responses obtained with RAW264.7 cells and the whole blood assay (WBA), suggesting that compounds demonstrating efficacy in RAW264.7 cells may be ineffective in humans, and, more importantly, compounds that are effective in humans may be missed with a RAW264.7 screen. Interestingly, there was also no significant correlation between the WBA and human PBMCs when the latter were cultured with 10% FCS, but a moderate correlation was seen when the PBMCs were cultured with 25% autologous plasma. The presence of plasma thus contributes to the overall inflammatory response observed in the WBA. We then asked if RAW264.7 cells, given that they are mouse macrophage-like cells, respond in a manner similar to primary murine derived macrophages. Intriguingly, there was no significant correlation (R=0.012) with the 37 putative immunomodulators, pointing to distinct inflammatory response mechanisms in the two model systems. We conclude that the use of a WBA to confirm potential immunomodulators obtained from high throughput screening with RAW264.7 cells is advisable and that future screens be carried out using a WBA.
RAW264.7小鼠巨噬细胞系被广泛用于进行免疫调节剂的体外筛选。能够有效降低脂多糖（LPS）刺激的RAW264.7细胞中促炎细胞因子或一氧化氮（NO）表达的化合物通常被视为人类抗炎药物的候选物。然而，关于该筛选方法识别真正人类免疫调节剂的可靠性的数据非常少。我们比较了37种所谓的免疫调节剂在RAW264.7细胞、全血和人外周血单核细胞（PBMC）实验中调节LPS或大肠杆菌诱导的炎症反应的效果。有趣的是，RAW264.7细胞实验和全血实验（WBA）得到的结果之间没有显著相关性（R = 0.315），这表明在RAW264.7细胞中显示出效果的化合物在人类中可能无效，更重要的是，在人类中有效的化合物可能会被RAW264.7筛选遗漏。有趣的是，当PBMC与10%胎牛血清（FCS）一起培养时，WBA和人PBMC之间也没有显著相关性，但当PBMC与25%自体血浆一起培养时，观察到中等程度的相关性。因此，血浆的存在有助于在WBA中观察到的整体炎症反应。然后我们询问，鉴于RAW264.7细胞是小鼠巨噬细胞样细胞，它们的反应方式是否与原代小鼠来源的巨噬细胞相似。有趣的是，与这37种假定的免疫调节剂没有显著相关性（R = 0.012），这表明两个模型系统中的炎症反应机制不同。我们得出结论，建议使用WBA来确认从RAW264.7细胞高通量筛选中获得的潜在免疫调节剂，并且未来的筛选应使用WBA进行。
