Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis.

OBJECTIVE: To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers. SAMPLE POPULATION: Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis. PROCEDURE: Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% alpha1-proteinase inhibitor (PI), 0.5% alpha1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples. RESULTS: Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% alpha1-PI, 93.6% by 0.5% alpha1-PI, and 90.0% by ES. CONCLUSIONS AND CLINICAL RELEVANCE: We documented that EDTA, doxycycline, NAC, ilomostat, alpha1PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses.

Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis.

PURPOSE: To study levels and activity of matrix metalloproteinase (MMP)-1 and -9 and their tissue inhibitor (TIMP-1) in tears of patients with vernal keratoconjunctivitis (VKC), with and without severe corneal damage. METHODS: Tear samples were obtained from 16 patients with active VKC and 10 normal control subjects, after clinical evaluation and tear cytology. Tear levels of pro-MMP-1, pro-MMP-9, and TIMP-1 were measured by enzyme-linked immunosorbent assay (ELISA). Collagenase and gelatinase activity were measured in tears by MMP activity assays. Immunohistochemistry was performed on a fragment of superficial keratectomy from two vernal corneal ulcers. RESULTS: Tear levels of pro-MMP-1 and pro-MMP-9 were significantly increased in patients with VKC compared with control subjects (P < 0.001). MMP-1/TIMP-1 and MMP-9/TIMP-1 molar ratios were significantly increased (P < 0.001) in VKC. MMP-1 and MMP-9 activities were significantly increased in VKC tears compared with control samples (P < 0.005). MMP-9 activity correlated significantly with corneal involvement and giant papillae formation. Immunohistochemistry showed positive staining for MMP-9, fibronectin, and eosinophil cationic protein (ECP) on the superficial corneal stroma of the ulcer bed, but no inflammatory cells. CONCLUSIONS: Increased levels and activity of MMP-1 and -9 and an imbalance between MMPs and TIMP may be involved in the pathogenesis of VKC.

Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro.

The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines. Corneal lesions and MMP induction in vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa. Effects of pseudomonal virulence factors [elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)], tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting. Corneal ulcers with typical ring abscesses were observed 12-24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas. Pseudomonal elastase caused the most extensive damage to both cell types. RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9. Exotoxin A had no effect on MMP expression. Both IL-1beta and TNF-alpha augmented MMP-9 expression in HT1080 cells. Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells. In conclusion, corneal destruction seen with P. aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase.

Effect of vitamin A deficiency on the early response to experimental Pseudomonas keratitis.

PURPOSE: Vitamin A-deficient humans and animals are more susceptible to infections than are healthy humans and animals. This study compares the early corneal response (within 24 hours) to an experimental Pseudomonas aeruginosa infection between vitamin A deficient and control rats. METHODS: Male WAG/Rij/MCW rats were fed either a vitamin A- deficient diet (A-) or the same diet with retinyl palmitate added back in a nonrestricted manner (N) or under pair-fed conditions (A+) to yield weight-matched rats. Some A-rats were repleted wih retinyl palmitate 16 days before being killed and then given free access to the retinyl palmitate-supplemented diet (R). Twenty-four hours before being killed, the corneas of anesthetized rats were scratched and P. aeruginosa organisms were applied to the corneal surface. The rats were killed using an overdose of sodium pentobarbital. Corneas were either processed for light and electron microscopic examination or extracted for proteinase and myeloperoxidase determination. Corneal myeloperoxidase concentrations relative to neutrophil myeloperoxidase concentrations were used to determine the number of neutrophils in the cornea. Zymography was used to study caseinases, gelatinases, and plasminogen activators. Reverse zymography was used to detect proteinase inhibitors. Similar results were noted at early, mid, and late weight plateau stages of vitamin A deficiency. RESULTS: Ulceration occurred within 24 hours when low numbers of P. aeruginosa (10(4) cpu) were applied topically onto scratched A- corneas, whereas no ulceration was observed in the A+, R, and N corneas. When higher numbers of P. aeruginosa (10(7)-10(8)) were applied to the scratched corneas, all corneas became ulcerated within 24 hours. The extent of ulceration in the control corneas was greater than that in A- corneas by a factor of two. Only the A- corneas contained inflammatory cells with unusual striated deposits in phagolysosomes. The total number of neutrophils in the cornea and the concentrations of caseinases, plasminogen activators, and gelatinases in the infected corneal extracts were similar; however, the concentrations of cysteine proteinase inhibitors were elevated under A- conditions. CONCLUSIONS: Vitamin A deficiency alters the response of the cornea to a P. aeruginosa infection during the first 24 hours. The alterations observed are probably due to multiple factors: an insufficient tear film for bacterial clearance and migration of neutrophils, epithelial keratinization, alterations in corneal wound healing, and changes in polymorphonuclear function.

Collagenase (MMP-1) and TIMP-1 in destructive corneal disease associated with rheumatoid arthritis.

The aim of the study was to immunolocalise interstitial collagenase (MMP-1) and the tissue inhibitor of metalloproteinases (TIMP-1) in ulcerating corneas from patients with rheumatoid arthritis, to determine whether changes in expression are associated with destructive corneal disease. Collagenase was expressed by stromal cells in 8 of 8 ulcerating corneas but was not seen in normal tissue (n = 3). TIMP-1 was abundant throughout the normal stroma, but was much reduced or absent from diseased corneas. Collagenase staining was frequently more intense near the epithelial surface and associated with a cellular infiltrate consisting of activated antigen-presenting cells (HLA-DR+), many of which were macrophages (CD68+) and derived from the epithelium or limbus (S100+). Interstitial collagenase produced by infiltrating macrophages and/or stimulated corneal fibrocytes is probably a major mediator of collagen degradation in rheumatoid corneal ulceration. In addition, reduced levels of TIMP-1 expression are consistent with collagenase activity and tissue destruction. Epithelial-stromal cell interactions and the production of local inflammatory mediators are of major importance in the pathogenesis of corneal destruction, although the precise nature of the antigenic stimulation and/or cellular interactions remains to be elucidated.

Development of monoclonal antibodies recognizing collagenase from rabbit PMN; the presence of this enzyme in ulcerating corneas.

Rabbit uterine collagenase was purified from the medium of involuting uterus (1-2 days postpartum) in culture using ammonium sulfate fractionation, DEAE-cellulose, heparin-affinity, and high performance liquid chromatography. The enzyme was purified more than 1600 fold. Hybridoma cell-lines producing monoclonal antibodies were prepared by fusing the spleen cells of mice immunized with the purified enzyme with mouse myeloma cells (Sp2/O-Ag14). The hybridoma cells were selected with HAT medium, cloned, and screened by ELISA. Antibody-producing ascites were prepared by injecting hybridoma cell-lines into the peritoneal cavities of mice. Western-blot analysis indicated that the antibodies recognized a polypeptide having a molecular weight of 52,000. The IgG isolated from the ascites inhibited the enzyme. Indirect immunofluorescent staining demonstrated that polymorphonuclear leukocytes (PMNs) in the superficial layer of alkali-burned corneas contained collagenase, whereas stromal cells and PMNs within the stroma were not stained by the antibodies. Our results suggest that collagenases produced by rabbit PMNs are different from those produced by fibroblasts from cornea. We hypothesize that PMNs in alkali-burned corneas secrete all or most of their collagenases by degranulation at the anterior surface of the cornea, and then continue to migrate into the deeper portion of the stroma.

Stromal vascularization prevents corneal ulceration.

Experiments were performed with a model of focal, thermal-induced ulceration to test the clinical impression that vascularization prevents ulceration of the corneal stroma. Slow-release polymers containing a vasoproliferase agent (tumor angiogenesis factor) were placed in corneal pockets 2 mm central to the limbus of albino rabbits. These polymers elicited blood vessel ingrowth up to the implant. Control eyes received empty polymers which caused minimal to no vessel growth. Polymers were removed, and each cornea received a focal, thermal burn placed just central to the polymer site. All control corneas ulcerated: most (79%) developed deep stromal or perforating ulcers. Only 25% of prevascularized corneas developed stromal ulcers, and none was deep or perforating. After thermal burns, vessels in both groups grew at the same linear rate toward the burned area. There was a direct relationship between the distance separating the nearest blood vessel and the burned area at the time of burning and the maximum depth of stromal ulceration. Thus prevention of or less severe stromal ulceration is correlated with the earlier presence of vessels in the burned area.

Collagenase in human cornea: immunologic localization.

Collagenase has been localized in human corneas using immunocytochemical techniques employing a specific antiserum to human skin collagenase. The enzyme was found in vivo in ulcerating corneas, whereas in corneas obtained from a variety of nonulcerative conditions, no immunologic evidence of the enzyme was seen. Corneal collagenase was present only in the stromal portion of the cornea, suggesting that cells from this tissue are the source of the enzyme. The localization of collagenase to the corneal stroma was reproduced by placing either ulcerating or nonulcerating corneas in tissue culture, indicating that the organ culture system may represent a model that duplicates some conditions present in corneal disease.