Gelatinase B/matrix metalloproteinase-9 is a phase-specific effector molecule, independent from Fas, in experimental autoimmune encephalomyelitis

Gelatinase B/matrix metalloproteinase-9 (MMP-9) triggers multiple sclerosis (MS) and the animal model of experimental autoimmune encephalomyelitis (EAE) by the breakdown of the blood-brain barrier. Interestingly, MMP-9 is beneficial in systemic autoimmunity caused by Fas-deficiency. Fas-deficient (faslpr) and Fas-ligand-deficient mice are protected against EAE. We here investigated the interaction between Fas and MMP-9 in the setting of induction of EAE and compared short- and long-term effects. We provoked EAE with myelin oligodendrocyte glycoprotein (MOG) peptide and compared EAE development in four genotypes (wild-type (WT), single knockout mmp-9-/-, faslpr, and mmp-9-/-/faslpr) and monitored leukocytes, cytokines and chemokines as immunological parameters. As expected, faslpr mice were resistant against EAE induction, whereas MMP-9 single knockout mice were not. In the double mmp-9-/-/ faslpr mice the effects on disease scores pointed to independent rather than interrelated disease mechanisms. On a short term, after EAE induction leukocytes infiltrated into the brain and cytokine and chemokine levels were significantly higher in all the four genotypes studied, even in the faslpr and mmp-9-/-/faslpr, which did not develop clinical disease. The levels of MMP-9 but not of MMP-2 were increased in the brain and in the peripheral organs after EAE induction. After 40 days all the animals recovered and did not show signs of EAE. However, the absence of MMP-9 in the remission phase suggested a protective role of MMP-9 in the late phase of the disease, because single mmp-9-/- mice presented a delayed remission in comparison with WT animals suggesting a phase-dependent role of MMP-9 in the disease. Nevertheless, the levels of some cytokines and chemokines remained higher than in control animals even 100 days after EAE induction, attesting to a prolonged state of immune activation. We thus yielded new insights and useful markers to monitor this activated immune status. Furthermore, MMP-9 but not MMP-2 levels remained increased in the brains and, to a higher extend, in the spleens of the WT mice even during the remission phase, which is in line with the role of MMP-9 as a useful marker and a protective factor for EAE in the remission phase.

明胶酶B/基质金属蛋白酶-9（Gelatinase B/matrix metalloproteinase-9, MMP-9）可通过破坏血脑屏障，诱发多发性硬化（multiple sclerosis, MS）以及实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）。值得注意的是，MMP-9在Fas缺陷（Fas deficiency）引发的全身性自身免疫病中却具有有益作用。Fas缺陷型（faslpr）小鼠与Fas配体缺陷（Fas-ligand-deficient）型小鼠可免受EAE侵袭。本研究探究了EAE诱导过程中Fas与MMP-9的相互作用，并比较了该过程的短期与长期效应。我们采用髓鞘少突胶质细胞糖蛋白（myelin oligodendrocyte glycoprotein, MOG）多肽诱导EAE模型，对4种基因型小鼠（野生型（wild-type, WT）、单基因敲除mmp-9-/-、faslpr以及mmp-9-/-/faslpr双敲除小鼠）的EAE发病情况进行比较，并以白细胞、细胞因子与趋化因子作为免疫学监测指标。正如预期，faslpr小鼠对EAE诱导具有抗性，而MMP-9单基因敲除小鼠则无此保护效应。在mmp-9-/-/faslpr双敲除小鼠中，疾病评分的变化提示其发病机制彼此独立，而非相互关联。在EAE诱导后的短期阶段，白细胞浸润至脑组织，且4种受试基因型小鼠的细胞因子与趋化因子水平均显著升高，即便在未出现临床疾病的faslpr小鼠与mmp-9-/-/faslpr双敲除小鼠中亦是如此。EAE诱导后，小鼠脑组织与外周器官中的MMP-9水平（而非MMP-2）出现显著升高。诱导40天后，所有受试小鼠均恢复健康，未表现出任何EAE相关临床症状。然而，缓解期内MMP-9的缺失提示其在疾病晚期发挥保护作用：与野生型小鼠相比，mmp-9-/-单基因敲除小鼠的缓解进程显著延迟，这表明MMP-9在疾病进程中发挥阶段依赖性的调控作用。尽管如此，即便在EAE诱导100天后，部分细胞因子与趋化因子的水平仍高于对照组小鼠，证实机体存在持续的免疫激活状态。本研究因此为监测该免疫激活状态提供了全新的见解与实用的生物标志物。此外，即便处于缓解期，野生型小鼠的脑组织中MMP-9水平（而非MMP-2）仍维持升高，且在脾脏中的升高幅度更为显著，这与MMP-9作为EAE缓解期有效标志物与保护因子的作用相符。