Increased expression of matrix metalloproteinases in vivo in scleritis tissue and in vitro in cultured human scleral fibroblasts.
Scleritis is a sight-threatening inflammatory disorder of the eye characterized by the degradation of scleral matrix. Matrix metalloproteinases (MMPs) are ubiquitous proteolytic enzymes important in physiological and pathological processes, the activity of which is stringently controlled by the action of a family of natural antagonists, the tissue inhibitors of matrix metalloproteinases (TIMPs). We hypothesized that enhanced expression of MMPs, without the negative regulatory influence of TIMPs, may be a key feature of tissue destruction in inflammatory eye diseases, such as scleritis. The aim of this study was to localize and characterize cells expressing MMPs and TIMPs in sclera affected by necrotizing scleritis and, in a parallel study, to establish whether cytokines modulate MMP expression in cultured human scleral fibroblasts. In situ hybridization and immunohistochemical analyses indicated that resident scleral fibroblasts as well as inflammatory cells such as macrophages and T lymphocytes express stromelysin, gelatinase B, and TIMP-1 in necrotizing scleritis tissue. In addition, cytoplasmic immunoreactivity for tumor necrosis factor-alpha, an inducer of MMPs, was detected in infiltrating inflammatory cells. Cultured scleral fibroblasts stimulated with the combination of interleukin-1 alpha plus tumor necrosis factor-alpha increased TIMP-1 mRNA twofold above constitutive levels. By contrast, these cytokines induced a sevenfold increase in the steady-state levels of stromelysin mRNA. Using Western blotting, stromelysin and TIMP-1 protein production paralleled mRNA induction in cytokine-stimulated human scleral fibroblasts. Culture supernatants harvested from cytokine-stimulated human scleral fibroblasts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis gelatin substrate zymography. Our results revealed a prominent 92-kd gelatinolytic band corresponding to gelatinase B, which was inducible with interleukin-1 alpha. These data provide evidence for our hypothesis, that an imbalance between enzyme/inhibitor ratios may be the underlying mechanism of the tissue destruction characteristic of scleritis. Our results demonstrate the potential involvement of MMPs and their modulation by cytokines produced by infiltrating inflammatory cells in destructive ocular inflammation.
Di Girolamo
N
N
Inflammation Research Unit, School of Pathology, University of New South Wales, Sydney, Australia.
Lloyd
A
A
McCluskey
P
P
Filipic
M
M
Wakefield
D
D
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008297:Male; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000378:metabolism*; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D012590:Sclera / Q000166:cytology / Q000378:metabolism*; D015423:Scleritis / Q000378:metabolism* / Q000473:pathology; D014018:Tissue Distribution; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D006023:Glycoproteins; D007375:Interleukin-1; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha; D017364:Collagenases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9