Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA.
J Neuroinflammation. 2011 Jun 24;8:73. doi: 10.1186/1742-2094-8-73.
Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 μg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.
实验性自身免疫性脑脊髓炎（EAE）是一种与中枢神经系统（CNS）脱髓鞘炎症性疾病人类多发性硬化（MS）相关的动物模型。通过过继转移诱导 EAE 可研究供体 T 淋巴细胞在疾病发病机制中的作用。可靠地诱导 C57BL/6（H-2b）小鼠过继转移 EAE 一直具有挑战性。本研究的目的是开发一种可重现且高产量的 C57BL/6 小鼠过继转移 EAE 方案。逐步的实验方法使我们能够开发出一种方案，该方案导致受者小鼠的疾病发生率约为 70%。供体小鼠用髓鞘少突胶质细胞糖蛋白（MOG）p35-55 在完全弗氏佐剂（CFA）中免疫，然后用百日咳毒素（PT）处理。只有在免疫后第 12 天分离的淋巴结细胞（LNC），并在体外用 10μg/mL 的 MOGp35-55 和 0.5ng/mL 的白细胞介素-12（IL-12）再刺激 72 小时，才能转移疾病。LNC 转移疾病的能力与第 12 天 CNS 中炎症浸润的存在有关。在免疫后第 6 天和第 12 天制备的细胞培养物中，IFNγ 的产生水平相当。相比之下，在我们的 EAE 模型中，IL-17 与疾病易感性呈负相关趋势。与免疫后第 6 天相比，在免疫后第 12 天收集的细胞培养物上清液中 GM-CSF 的分泌量显著增加。在第 12 天 LNC 培养物中活化的 CD4+T 细胞维持转录因子 T-bet 的表达，该转录因子已被证明可调节 IL-23 受体的表达。此外，在体外再刺激后，第 12 天 LNC 中存在更多的 MOGp35-55 特异性 CD4+T 细胞。总之，在我们的研究中，过继转移 C57BL/6 小鼠 EAE 的致脑炎性 LNC 并没有单一的生物标志物来表征，而是由一组炎症标志物来表征。我们的数据进一步表明，由 IL-23 调节的 CD4+T 细胞表达的 GM-CSF 有助于我们的 EAE 模型中它们的致脑炎性。
