The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention

Abstract

Background: Extracellular proteases such as plasminogen activators (PAs) and matrix metalloproteinases modulate cell-cell and cell-matrix interactions. Components of the PA/plasmin system have been shown to be increased in areas of inflammation, and have been suggested to play a role in inflammatory neurologic disorders such as epilepsy, stroke, brain trauma, Alzheimer's' disease and multiple sclerosis (MS). In the present study, we evaluated the involvement of the PA system in the animal model of MS, experimental autoimmune encephalomyelitis (EAE).

Methods: EAE was induced by myelin oligodendrocyte glycoprotein (MOG) in mice deficient for the urokinase PA (uPA-/-), or the urokinase PA receptor (uPAR-/-). Mice were evaluated for EAE clinical signs and histopathologic parameters, and compared with wild-type (WT) EAE mice. Lymphocytes from the knockout (KO) and WT mice were analyzed for ex vivo restimulation, cytokine secretion, and antigen presentation. Finally, WT EAE mice were treated with PAI-1dp, an 18 amino acid peptide derived from the PA inhibitor protein (PAI-1).

Results: EAE was aggravated in uPA-/- and uPAR-/- mice, and this was accompanied by more severe histopathologic features and microglial activation. By contrast, specific T- cell reactivity towards the encephalitogenic antigen MOG was markedly reduced in the KO animals, as shown by a marked reduction in proliferation and pro-inflammatory cytokine secretion in these mice. Antigen presentation was also reduced in all the KO animals, raising an immunologic paradox. When the mice were treated with PAI-1, a peptide derived from the PA system, a marked and significant improvement in EAE was seen. The clinical improvement was linked to reduced T-cell reactivity, further emphasizing the importance of the PA system in immunomodulation during neuroinflammation.

Conclusions: Cumulatively, our results suggest a role for uPA and uPAR in EAE pathogenesis, as exacerbation of disease was seen in their absence. Furthermore, the successful amelioration of EAE by PAI-1 treatment suggests that the PA system can be considered a potential site for therapeutic intervention in the treatment of neuroimmune diseases.

Figure 1

Experimental autoimmune encephalomyelitis (EAE) is aggravated in mice deficient for the urokinase plasminogen activator (uPA^−/−), or the urokinase plasminogen activator receptor (uPAR^−/−). (A) EAE clinical severity in uPA^−/− mice and control wild-type (WT) mice. (B) EAE clinical severity in uPAR^−/− mice and control WT mice. (A,B) Results are expressed as the mean clinical score ± standard error (SE) of three separate experiments (*P<0.05).

Figure 2

Neuropathology in the spinal cords of EAE in urokinase plasminogen activator (uPA^−/−), or the urokinase plasminogen activator receptor (uPAR^−/−) mice compared with WT mice. The results are presented as mean ± standard error (SE) (infiltrates/mm²). (A) uPA^−/− animals exhibited an almost twofold greater inflammatory burden (**P<0.001). (a1,a2) Representative photographs (hematoxylin plus Bielschowsky stain) Magnification x200. (B) uPAR^−/− animals exhibited significantly greater inflammation (*P<0.05). (b1,b2) Representative photographs, magnification x200.

Figure 3

Axonal injury and axonal loss in experimental autoimmune encephalomyelitis (EAE) in mice deficient for the urokinase plasminogen activator (uPA^−/−), or the urokinase plasminogen activator receptor (uPAR^−/−) compared with EAE wild-type (WT) mice. Rsults are presented as percentage distribution of severity. (A,C) uPAR^−/− mice exhibited (A) similarly active axonal injury (AI) and (C) significantly greater axonal loss (AL). (B,D) uPA^−/− mice exhibited (B) more severe AI and (D) more AL. *P<0.05, **P<0.001.

Figure 4

Lectin-positive microglia/macrophages in spinal cords of mice deficient for the urokinase plasminogen activator receptor (uPAR^−/−) and of wild-type (WT) mice. (A) Significant increase in numbers of lectin-positive cells (microglia/macrophages) in the spinal cords of uPAR^−/− animals compared with the WT mice (***P<0.0001). Representative photographs showing perivascular and parenchymal lectin-positive microglia/macrophages (arrows) in (B,C) WT and (D,E) uPAR^−/− animals.

Figure 5

mice deficient for the urokinase plasminogen activator (uPA^−/−), or the urokinase plasminogen activator receptor (uPAR^−/−) show reduced T-cell reactivity to myelin oligodendrocyte glycoprotein (MOG)_35–55 and reduced antigen presentation. (A) Proliferation of lymphocyte from all the tested groups was checked by [³H] thymidine incorporation in the presence of MOG_35-55. (B). The effect of uPA and uPAR deficiency on T helper 1 (Th1) cytokine secretion. Lymphocytes from uPA^−/−, uPAR^−/− and WT mice were stimulated with MOG_35-55, the media were collected after 24 hours, and the secreted (B) interferon (IFN)-γ and (C) tumor necrosis factor (TNF)-α were measured by ELISA. (A–C) Results are expressed as mean ± standard error (SE) of three separate experiments (*P<0.05) (D) Effect of uPA and uPAR deficiency on antigen presentation measured by MOG_35-55 specific T-cell proliferation. Lymphocyte proliferation was checked by [³H] thymidine incorporation using APCs from knockout (KO) or WT mice in the presence of MOG_35-55. Results are expressed as the mean ± SE of four separate experiments (*P<0.05).

Figure 6

Administration of the plasminogen activator inhibitor-derived peptide PAI-1dp ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE). EAE was induced in wild-tupe (WT) mice by immunization with myelin oligodendrocyte glycoprotein (MOG)_35–55. The animals were given either placebo or PAI-dp 0.5 mg/kg twice daily by intraperitoneal injection. Results are expressed as the mean clinical score ± standard error (SE) of three separate experiments. (n = 16 for each group). The results are the mean of three separate experiments.

Figure 7

Administration of PAI-1dp reduces T-cell reactivity towards MOG_35-55 peptide. A. PAI-1dp pre-treatment reduced T cell proliferation induced by MOG_35-55. B. Reduced secretion of the pro-inflammatory cytokines IFN-γ and IL-17. The results are the mean standard error (SE) of three separate experiments (*P<0.05).