Expression and localization of cytokines and proteases in experimental pseudomonal keratitis

Abstract

Abstract: : Purpose: To investigate the expression and localization of cytokines and proteases in experimental pseudomonal keratitis. Methods:A clinical isolate of Pseudomonas aeruginosa (2x10⁴CFU) was injected into the right eyes of 32 New–Zealand albino rabbits and the eyes were enucleated at 3,9,12,15,18,24 and 72 hours post injection and processed to immunostaining for interleukin (IL)–1ß, tumor necrosis factor (TNF)–α, transforming growth factor (TGF)–ß, matrix metalloproteinase (MMP)–2 and MMP–9. Mouse–anti human cytokines monoclonal antibodies were used as primary antibodies. Furthermore, corneas were excised and the extracts were processed for gelatin– and casein– zymography to detect proteases such as MMPs. Results:Corneal ulcers with ring abscesses were observed 12–15 hours postinfection, and progressed during the infection. Immunohistochemical studies demonstrated that IL–1ß, TNF–α and TGF–ß were mildly expressed in the corneas around the injection site in the early phase of infection, whereas enhanced immunostainings were observed for these cytokines after abscess formed. MMP–2 was immunostained in the corneas even in the early phase of infection. However, an enhanced immunostaining was not observed even though the progression of the corneal lesion. In contrast, MMP–9 was immunostained significantly especially in abscess site. Gelatin–zymography showed that MMP–2 was expressed in the cornea from early phase of infection, but its activity was gradually reduced 15–72 hours postinfection. In contrast , MMP–9 was firstly detected at 12 hours postinfection and active form of MMP–9 as well as pro–MMP–9 were significantly enhanced 24 – 72 hours postinfection. Casein–zymography showed the presence of caseinase with an approximate molecular weight of 25 kDa 18 hours post infection. Conclusions:Because the significant expressions of IL–1ß, TNF–α, TGF–ß, MMP–9 and caseinase were observed during corneal ulcer formation in experimental pseudomonal keratitis, it is indicated that these factors may contribute to the corneal ulcer formation.

Keywords: keratitis • cytokines/chemokines • Pseudomonas