The data presented in this study show that the PA system plays a role in CNS inflammation. Using mice lacking uPA and uPAR, we found that during EAE, mice exhibit a very severe disease, with impaired recovery. The neuropathologic findings were consistent with disease severity; uPA−/− and uPAR−/− mice showed an increase in AL and a massive infiltration of mononuclear cells into the spinal cord.
In addition, the importance of microglial activation in EAE pathology is known, and we found that microglial activation was increased twofold within the spinal cord of uPAR−/− mice. Microglia are resident APCs that make up to 10 - 20% of all the glial cells in the CNS . In MS and EAE, microglia are activated, express high levels of major histocompatibility complex class II, and function as APCs [20, 21]. The increase in lectin-positive microglia/macrophage activation seen in the uPAR−/− animals is in accordance with the clinical severity of the disease.
Our results are in agreement with previously reported results. East et al. reported a more severe EAE in tPA−/− mice, characterized by incomplete recovery and increased neurologic deficit, and also reported persisting inflammatory cuffs of mononuclear cells and a greater degree of demyelination in uPAR−/− mice . Another report by East et al. showed that mice lacking the plasminogen inhibitor PAI-1 developed milder EAE without clinical relapse, and with an overall reduction in neuroinflammation .
These findings, together with those from the earlier part of the current study, prompted us to attempt to treat EAE using the PAI-dp, the peptide derived from the PAI-1 protein. PAI-1dp inhibits the action of PAI-1, thereby prolonging the presence and proteolytic activity of uPA. Importantly, we found that when EAE mice were pre-treated with PAI-1dp, they developed a markedly less severe disease, accompanied by a reduction in T-cell reactivity. To our knowledge, this amelioration of EAE after the novel approach of injecting peptides derived from the PA system has not been reported previously.
The roles played by the PA system in the pathogenesis of EAE could be ascribed to a number of different mechanisms, such as regulation of fibrin deposition at sites of inflammation, and effects on cell trafficking into the CNS. Fibrin, the end product of PA proteolytic activity, participates in a variety of cellular responses associated with inflammation [23, 24] by binding to a range of receptors that are expressed on leucocytes, macrophages, and monocytes . Fibrinogen-deficient mice show delayed inflammatory responses to lipopolysaccharide . Because fibrin is capable of modulating inflammation via a number of different mechanisms, it is clear that removal of fibrin by enhancing fibrinolysis can produce beneficial results.
Although the uPA−/− and uPAR−/− mice exhibited more severe disease, there was a reduction in their immune response compared with WT mice. This was reflected by a reduction in proliferative lymphocytes and a reduced ability to present antigen. These findings point to a paradoxical situation in which the KO mice present with more severe disease, a condition that is usually characterized by high lymphocyte proliferation and cytokine induction, but in this study showed just the opposite results. The PA system is considered to be both pro-inflammatory and anti-inflammatory. The former is related to the mounting of correct innate and adaptive immune responses, whereas the latter is related to prevention of extracellular fibrin deposition . The PA system components involvement in two opposing processes might explain these conflicting results.
There is an intriguing link between the uPA/uPAR system and the immune system. Both uPA and uPAR expression were shown to be modulated by inflammatory mediators. TNF-α, IFN-γ, and IL-1 were found to increase uPA expression on macrophages [27, 28]. Activated T lymphocytes express uPA and uPAR [29, 30], and stimulation with phorbol esters and T-cell receptor (TCR)-mediated stimulation results in substantial upregulation of uPA and uPAR in T cells . uPAR is co-expressed with CD25 (IL-2R), and its expression in TCR-mediated T-cell activation has been established . Furthermore, uPAR is upregulated by exposure to IL-2 and IL-4, but not to several other cytokines . Conversely, there is substantial evidence suggesting that uPA is a modulator of immune and inflammatory responses. For example, early reports showed that PA could act as a lymphocyte mitogen . Thus, bidirectional communication links the uPA/uPAR system and the inflammatory cytokine networks. Therefore, the profound effect of uPA/uPAR deficiency on lymphocyte activation in our model is not unexpected, and the severe clinical outcome can be explained by the inability of the KO mice to mount a protective immune response. Similar results were reported by Gyetko et al.  who investigated the ability of uPA−/− mice to mount a protective host defense during infection with the opportunistic yeast Cryptococcus neoformans. In the absence of uPA, the mice failed to mount an adequate immune defense against the yeast, resulting in a lethal defect in cell-mediated immune responses.
When targeting the PA system for treatment of EAE/MS, it is important to bear in mind its major role in the fibrinolytic system, and that manipulation of the PA system might cause unwanted effects in the coagulation/fibrinolytic system. Recombinant tPA has been in clinical use for treatment of myocardial infarct. However, it can be given only once in such cases, and not as an ongoing treatment because it causes excessive bleeding, and therefore cannot be used for treatment of MS.
It is interesting that mice lacking uPA do not have major thrombotic disorders . This is most probably because of the redundant fibrinolytic function of uPA, which, at least in the vascular compartment, can be substituted for by tPA. Double uPA-tPA KO mice show extensive thrombotic disorders similar to those in plasminogen KO mice . In addition, disruption of the PAI-I gene in mice does not appear to impair hemostasis, but is associated with increased resistance to thrombosis and with a mild hyperfibrinolytic state characterized by enhanced in vivo clot lysis . Interestingly, despite the minor influence of the disruption of these genes in the naïve state, the effects of disruption become apparent upon disease induction. This suggests that the biologic consequences of PA system gene inactivation might be more significant in disease states.