Upregulation of MMP-9/TIMP-1 enzymatic system in eosinophilic meningitis caused by Angiostrongylus cantonensis

Abstract

Proteolysis depends on the balance between the proteases and their inhibitors. Matrix metalloproteinase-9 (MMP-9) and its specific inhibitors, tissue inhibitors of metalloproteinases (TIMP), contribute to eosinophilic inflammatory reaction in the subarachnoid space of the Angiostrongylus cantonensis-infected mice. The expression of MMP-9 in cerebrospinal fluid (CSF) was significantly increased in mice with eosinophilic meningitis, compared to that in uninfected ones. However, the TIMP-1 levels were unchanged and remained at basal levels at all time points, even in uninfected mice. Elevated MMP-9 mRNA expression coincided with protein levels and proteolytic activity, as demonstrated by means of positive immunoreactivity and gelatin zymography. CSF protein contents correlated significantly with MMP-9 intensity and CSF eosinophilia. In addition, immunohistochemistry demonstrated MMP-9 and TIMP-1 localization in eosinophils and macrophages. When the specific MMP inhibitor, GM6001, was added, MMP-9 enzyme activity was reduced by 45.4%. The percentage of eosinophil increased significantly upon the establishment of infection, but subsided upon inhibition. These results show that MMP-9/TIMP-1 imbalance in angiostrongyliasis may be associated with eosinophilic meningitis.

Figure 1

Expression of MMP-9 and TIMP-1 mRNA in the CSF. (a) The MMP-9 mRNA was upregulated on day 10 post-inoculation, showing a high expression from day 15 to 25 post-inoculation. TIMP-1 mRNA was unchanged and remained at basal levels at all time points, even in uninfected mice. GAPDH mRNA was performed for a loading control. (b) Quantitative analysis of MMP-9 and TIMP-1 mRNA was performed with a computer-assisted imaging densitometer system. Ratios of MMP-9/TIMP-1 showed statistically significant elevation (* P < 0.05) on days 10, 15, 20 and 25 post-inoculation. CSF, cerebrospinal fluid; MMP-9, matrix metalloproteinase-9; TIMP, tissue inhibitors of metalloproteinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Matrix metalloproteinase-9 (MMP-9) and TIMP-1 protein levels in the cerebrospinal fluid (CSF). (a) The 94 kDa MMP-9 was present on days 15, 20 and 25 post-inoculation. The 28 kDa TIMP-1 was unchanged and remained at basal levels at all time points, even in those derived from uninfected mice. β-Actin was used as a loading control. (b) Quantitative analysis of MMP-9 and TIMP-1 was performed with a computer-assisted imaging densitometer system. Ratios of MMP-9/TIMP-1 showed statistically significant elevation (* P < 0.05) on days 15, 20 and 25 post-inoculation. TIMP, tissue inhibitors of metalloproteinases.

Figure 3

Correlation of matrix metalloproteinase-9 (MMP-9) activity with cerebrospinal fluid (CSF) eosinophils. (a) The time-course studies showed that MMP-9 activity was detected on day 10 post-inoculation and reached a high intensity from day 15 to 25 post-inoculation. (b) Quantitative analysis of the gelatinolytic enzyme was performed with a computer-assisted imaging densitometer system. The relative intensity of MMP-9 activity showed statistically significant elevation (* P < 0.05) on days 10, 15, 20 and 25 post-inoculation. (c) The relative intensity of MMP-9 activity correlated significantly (* P < 0.05) with CSF eosinophil percentages by using Spearman's rank correlation test.

Figure 4

Correlation of cerebrospinal fluid (CSF) total protein with matrix metalloproteinase-9 (MMP-9) activity and eosinophil percentage. CSF protein contents correlated significantly with the MMP-9 intensity (a) and eosinophil percentage (b) by using the Spearman's ranking correlation test.

Figure 5

Histological and immunohistochemical observations in the subarachnoid space. (a) The brain showing young adult worm of Angiostrongylus cantonensis (arrowheads) invaded the subarachnoid space, and accompanied severe haemorrhage (H) and numerous leucocytes (L). C, cerebral cortex. Hematoxylin and eosin staining. (b) Enlargement of the portion showed in rectangle of (a).The leucocytes aggregation in the subarachnoid space was observed, and especially, eosinophilic (arrows) and neutrophilic (blank arrowheads) leucocytes infiltrated the oedema meninges. Black arrowhead indicates macrophage. (c) No matrix metalloproteinase-9 (MMP-9) and TIMP-1-positive signal could be detected with normal serum in polymorphonuclear and mononuclear cells, including eosinophils (arrows) or macrophages (arrowheads). (d) Detection with a polyclonal antiserum against MMP-9 showing MMP-9 localized in eosinophils (arrows) and macrophages (arrowheads). (e) TIMP-1-positive signal could be detected in eosinophils (arrows) and macrophages (arrowheads). TIMP, tissue inhibitors of metalloproteinases.

Figure 6

Effect of matrix metalloproteinase (MMP) inhibition on MMP-9 activity and eosinophilic meningitis. (a) The molecular weight of 94-kDa MMP-9 lytic area was reduced by 45.4%, compared with mice infected with Angiostrongylus cantonensis on day 20 post-inoculation. (b) Quantitative analysis of the gelatinolytic enzyme was performed with a computer-assisted imaging densitometer system. The relative intensity of MMP-9 activity in the inhibitory group was significantly decreased (*P < 0.05), compared to that in the infected group. (c) Eosinophil percentage significantly decreased (*P < 0.05) in GM6001-inhibited mice, compared to that in mice infected with A. cantonensis on day 20 post-inoculation. Control, uninfected mice; infected, A. cantonensis-infected-uninhibited mice; GM6001, GM6001-inhibited-infected mice.