Matrix metalloproteinases in recurrent corneal melting associated with primary Sjörgen's syndrome

Abstract

Purpose: To investigate the contribution of matrix metalloproteinases (MMPs) to recurrent corneal melting in keratoconjunctivitis sicca associated with primary Sjörgen's syndrome (pSS).

Methods: One native melted cornea and ten melted corneal grafts from two patients with severe pSS were used. The presence of MMPs (1, 2, 3, 7, 8, 9, and 13) was detected using indirect enzyme immunohistochemistry. The active forms of MMP 2 and 9 and MMP 3 and 7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP 1 were measured using an activity assay. Eleven unaffected corneas served as controls.

Results: The average values of the staining intensity revealed very intense MMP 1, intense MMP 2, 7, and 9 and moderate MMP 3 and 8 positivity, in the corneal epithelium of melted corneas. Intense MMP 1 and 9 staining, moderate MMP 2, 3, and 8 staining, and weak MMP 7 staining were found in the anterior stroma. The posterior stroma revealed intense MMP 1, moderate MMP 3 and 9, and weak MMP 2, 7, and 8 positivity. Immunostaining of the endothelium was moderate for MMP 9 and weak for MMP 1, 2, 3, 7, and 8. MMP 13 was negative in all but four melted specimens, where weak-to-moderate staining was found in the epithelium and stroma. Control corneas revealed moderate MMP 1 and 2 and weak MMP 8 staining in the epithelium, weak MMP 2 staining in the anterior stroma, and weak MMP 1 and 8 staining in the endothelium. Significantly elevated MMP 1 activity and extremely elevated MMP 9 activity were found in most of the tested pathological specimens, compared to healthy controls, where no activity of the two enzymes was present. Markedly elevated MMP 2 activity was found in 2 of 11 specimens, compared to normal tissue. The inactive form of MMP 3 was detected in half of the tested specimens, and inactive MMP 7 in all melted corneas. Active MMP 3 and 7 were found in one melted sample. Neither of these MMPs was found in any of the control specimens.

Conclusions: The increased expression and elevated activity of a wide range of MMPs in melted cornea samples from two patients diagnosed with pSS confirm that these enzymes contribute to the tissue destruction, leading to serious consequences such as corneal perforation and loss of vision.

Figure 1

Immunohistochemical localization of matrix metalloproteinases in melted and control corneas. Immunohistochemical localization of MMP 1, 2, 3, 7, 8, 9, and 13 in representative images of melted corneal specimens obtained from patients with pSS (cases 1 and 2) and from control corneas. Original magnification, 100×. Ep = epithelium, AS = anterior stroma, PS = posterior stroma, En = endothelium.

Figure 2

Gelatin zymography of matrix metalloproteinases 2 and 9 in melted and control corneas. Melted specimens (A) S1 and S2 showed extremely high levels, and specimens S7, S9, and S11 considerable levels, of both the proenzyme (67 KDa band) and the active form of MMP 2 (62 kDa band). Levels of MMP 9 proenzyme (95 kDa band) and the active form (82 kDa band) were extremely high in S4 and S6-S11, and prominent in S5. Weak bands for both MMP 9 forms were found in specimens S1-S3. In control samples (B), a moderate level of the MMP 2 proenzyme was present in all specimens, whereas the active form of MMP 2 was either not present or very faint, except in samples C1, C3, and C11. As for MMP 9 proenzyme, only C3 and C7 showed faint bands.

Figure 3

Casein zymography of matrix metalloproteinases 3 and 7 in melted specimens. Very slight bands of only the MMP 3 proform (59 kDa) were detected in five melted specimens (S1, S2, S6, S7, and S9) and of both the proform and active MMP 3 (47 kDa) in one specimen (S5). High levels of MMP 7 proenzyme (30 kDa) and its intermediate cleavage product (28 kDa) were detected in nine melted corneas, S2-S10, and weak bands of these two MMP 7 inactive forms were found in two melted specimens (S1 and S11). A very slight band of active MMP 7 (20 kDa) was found in one specimen only (S7).