Coordinated induction of extracellular proteolysis systems during experimental autoimmune encephalomyelitis in mice

Abstract

Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) are considered to play an important role in the pathogenesis of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is widely used as an animal model of multiple sclerosis. Whereas several studies have addressed the expression of various MMPs and their inhibitors in the pathogenesis of EAE, the expression of the molecules of the PA system during EAE has not been reported previously. The present study was undertaken to investigate the expression of the molecules of the PA system (tPA, uPA, PAI-1, uPAR, LRP), as well as several members of the MMP family and their inhibitors in the course of actively induced EAE in BALB/c mice. During clinical EAE, the PA system was up-regulated in the central nervous system at several levels. Induction of expression of tPA and PAI-1 transcripts was detected in activated astrocytes in the white matter. Inflammatory cells expressed uPA receptor, uPAR. In situ zymography demonstrated the presence of increased tPA and uPA activities in the areas of the inflammatory damage. Accumulation of fibrin, fibronectin, and vitronectin immunoreactivity was seen in perivascular matrices of symptomatic animals. In addition, transcription of MT1-MMP and metalloelastase (in inflammatory cells), and TIMP-1 (in activated astrocytes) was induced during EAE. Increased gelatinolytic activity was detected at the sites of inflammatory cell accumulation by in situ zymography of fluorescently labeled gelatin; substrate gel zymography identified the up-regulated gelatinolytic activity as gelatinase B. Overall, our study demonstrates concurrent induction of PA and MMP systems during active EAE, supporting further the concept that the neuroinflammatory damage in EAE involves altered balance between multiple extracellular proteases and their inhibitors.

Figure 1.

Expression of transcripts of tPA, PAI-1, uPAR, MT1-MMP, metalloelastase (MEL), and TIMP-1 in transversal sections of lumbar spinal cord of mouse with grade 3 EAE (columns 1 and 2) and lumbar spinal cord of control mouse (column 3). A to C: Bright-field images of luxol fast blue/cresyl violet-stained spinal cord sections demonstrate the presence of inflammatory cell deposits (arrowheads) at sites of immune cell accumulation in EAE (A and B) and not in nonsymptomatic spinal cord (C). Inset in B shows a higher magnification view with infiltrating perivascular inflammatory cells. During clinical EAE, tPA is up-regulated in scattered cells in the periphery of the white matter (D and E), whereas its expression in control spinal cord was under the same conditions below the detection limit (F). High levels of PAI-1 transcripts were detected in glial cells adjacent to the inflammatory lesions in spinal cord of symptomatic animals (G and H), whereas control CNS appeared negative (I). Prominent expression of transcripts of uPAR was observed in perivascular/meningeal inflammatory cells (J and K), whereas control spinal cord was negative for the expression of uPAR (L). MT1-MMP was similarly induced in the inflammatory cells (M and N), albeit at a lower level. Metalloelastase expression was not detectable in the control spinal cord (R); its expression was induced at EAE in a small subset of inflammatory cells (P and Q). Widespread expression of TIMP-1 transcripts was detected throughout the white matter of EAE spinal cord (S and T), whereas control spinal cord appeared negative for expression of TIMP-1 (U). Integrity of the RNA in the control tissue sections was demonstrated by positive hybridization signal for LRP, TIMP-2, and TIMP-3 (data not shown). Abbreviations: gt, gracile tract; dh, dorsal horn; wm, white matter; vh, ventral horn; vf, ventral (anterior) fissure. Scale bars: 220 μm (column 1); 45 μm (columns 2 and 3).

Figure 2.

Expression of tPA, PAI-1, and TIMP-1 genes in the pons of BALB/c mice with acute grade 4 EAE. A: Low magnification bright-field image of luxol fast blue/cresyl violet-stained section; dashed box indicates the region shown at higher magnification in microphotographs (C to E). C to E: tPA, PAI-1, and TIMP-1 are expressed by the scattered cells in transversal fibers of the white matter of the pons, with pontine nuclei scoring negative. B: Combined in situ hybridization analysis/glial fibrillary acidic protein immunostaining, demonstrating that tPA is expressed by hypertrophic astrocytes. Abbreviations: ft, fibrae transversae pontis; fl, fibrae longitudinales pontis; np, nuclei pontis; ml, crus cerebri; bp, basis pontis. Scale bars: 350 μm (A); 20 μm (B), 90 μm (C to E).

Figure 3.

Zymographic analysis of PAs in lumbar spinal cords of mice with grade 3 EAE and control mice. a: In situ zymography of PAs on cryosections of normal lumbar spinal cord (top row) and spinal cord of mice with grade 3 EAE. A: Control spinal cord sections exhibit a weak plasminogen-dependent caseinolytic activity in the area of dorsal root exit zone; B: the activity is not affected by inhibition of uPA. D: After the same time of incubation at 37°C, EAE spinal cord sections show strong caseinolysis in the periphery of the white matter, in particular in the gracile tract, along meninges and in the central canal area. E: Amiloride appears to block caseinolysis along meninges and reduce it in other locations. C and F: Omission of plasminogen in the overlay mixture abolishes caseinolysis in both control and EAE spinal cord. Scale bar, 280 μm. b: SDS-PAGE zymography of the PAs in lumbar spinal cord extracts of three untreated mice and three mice with grade 3 EAE. As tPA and uPA standards, urine from uPA and tPA null mice was used. Control spinal cords exhibited uPA, tPA, and plasmin activities. Caseinolytic activities corresponding to low molecular weight uPA (lmw uPA), plasmin and PA-inhibitors complexes (migrating between tPA and plasmin activity) appear elevated at EAE.

Figure 4.

Zymographic analysis of gelatinases in the lumbar spinal cord extract of the naïve mice and mice with grade 3 EAE. a: In situ zymography of gelatinases using DQ-gelatin. A and C: Increased green fluorescence indicating the cleavage of the DQ-gelatin is present in the areas of accumulation of inflammatory cells. B: Inhibition of metalloproteinases by 1,10-phenanthroline (PA) reduces the cleavage of the DQ-gelatin. C and E: Nuclear staining with 4.6-diamidino-2-phenylindole of the same section shown on A, B, and C. D: In control spinal cord sections fluorescence was observed occasionally over blood capillaries. Scale bar, 70 μm. B: SDS-PAGE zymography of gelatinases in the lumbar spinal cord extracts of three naïve mice and three symptomatic mice with grade 3 EAE. Note that whereas gelatinase A is constitutively expressed, gelatinase B is up-regulated during EAE. Upper high molecular weight complexes correspond to gelatinase B dimers.

Figure 5.

Localization of immunoreactivities of fibrin (A and B), fibronectin (C and D), and vitronectin (E and F) in the lumbar spinal cord of control mouse and of mouse with of grade 3 EAE. Note increased immunoreactivity in the perivascular matrix and along the meninges for all of the four antigens in EAE spinal cords (B, D, and F) and not in control spinal cords (A, C, and E). In contrast, laminin immunoreactivity remained unchanged (not shown). Scale bar, 280 μm.