Studies On The Mechanisms Of Collagen Degradation Of Corneal Ulcer

Corneal ulceration is frequently encountered and blindness disease in the ophthalmological area. Corneal ulceration results from the destruction of collagen fibrils in the corneal stroma. The corneal stroma comprises mostly type I collagen and resident fibroblats. fibroblasts are embedded in extracellular matrix (ECM) proteins, that these cells form an extensive and continuous network parallel to the plane of collagenous lamellae. Corneal ulceration is due to excessive melt of corneal collagen and glycoprotein. Activated orneal fibroblasts and infiltrated leukocytes are thought to play an important role in the pathobiolgy of corneal ulceration by contributing to the degradation of stromal collagen. Because they produced the enzymes to degrade the extracellular matrix. Corneal fibroblasts produced MMPs(matrix metalloproteinases), which were secreted extracellularly as pro-MMPs and activated by serine protease such as fibrinolysase, kallikrein and neutrophil elastase. cytokines regulate the expression of MMPs of corneal fibroblasts. Neutrocyte can defend an organism against bacterial and fungal infection,in the meanwhile lead to pathogenous injury by releasing hydrogen peroxide,free radicals, elastase, cathepsin,collagenase and gelatin. Neutrophil elastase can degrade many extracellular matrix protein such as elastin, glycoprotein, collagen, fibronectin, and participate many pathologic changes, for example, rheumatoid arthritis and bronchitis. But the role of neutrophil and corneal fibroblasts in corneal ulceration and its interaction with cytokines are not clear. The mechamisms of collagen degradation of corneal ulcer are not clear. Using three-dimensional collagen culture model of corneal fibroblasts and neutropils,To investigate the roles of interactions between resident fibroblasts and infiltrated neutrophils and between extracellular collagen and these cells in collagen degradation and corneal ulceration, we have now examined the effect of coculture of neutrophils with corneal fibroblasts on collagen degradation. We have also examined the effect of conditioned medium(CM) from neutrophils cultured either in three-dimensional collagen gels or as monolayers on fibroblast-mediated collagen breakdown.We have also examined the Effect of IL-1ra and dexamethasone on collagen degradation by cocultures of neutrophils and corneal fibroblasts.The collagen degradation were measured by the quantitation of hydroxyproline in the culture media,and the MMP activity were analyzed by Gelatin zymography and western blotting. The amounts of mRNA were determined by RT and real-time PCR analysis. RESULTS(1) We first examined collagen degradation in three-dimensional cultures of either corneal fibroblasts or neutrophils. Corneal fibroblasts mediated collagen degradation in a time-dependent manner. The amount of collagen degraded was significantly greater than that apparent in cell-free control cultures. In contrast, culture of neutrophils in collagen gels had no significant effect on collagen degradation. The effect of coculture of neutrophils and corneal fibroblasts on collagen degradation. Whereas neutrophils cultured in the absence of corneal fibroblasts had no significant effect on collagen degradation, the addition of neutrophils to corneal fibroblast cultures resulted in an increase in the extent of collagen degradation by fibroblasts that was dependent on the number of neutrophils added. ,although the extent of collagen degradation increased with cell number in cultures of fibroblasts alone, this effect was not statistically significant. Culture of neutrophils alone also had no effect on collagen degradation. However, the addition of fibroblasts to the neutrophil cultures resulted in an increase in the amount of collagen degraded that was significant and dependent on fibroblast number. Measurement of collagenolytic activity: The amount of degraded collagen in culture supernatants was expressed as micrograms of hydroxyproline.(2) we investigated the effect of neutrophil-conditioned medium (Ne-CM) on the collagenolytic activity of corneal fibroblasts. Neutrophil-CM from monolayer or collagen gel cultures alone did not affect the integrity of collagen. Conditioned medium from monolayer cultures of neutrophils induced an ~2.6-fold increase in the amount of collagen degraded by corneal fibroblasts. However, neutrophil-CM derived from collagen gel cultures induced an ~10-fold increase in collagen degradation by corneal fibroblasts. We also examined the effect of fibroblast-CM on collagen degradation by neutrophils. Whereas fibroblast-CM from monolayer cultures did not degrade collagen, that derived from collagen gel cultures mediated a low level of collagen degradation in the absence of neutrophils. However, the addition of either type of fibroblast-CM to collagen gel cultures of neutrophils did not increase the collagenolytic activity of these latter cells. Thus, although corneal fibroblasts also appeared to be activated by extracellular collagen, soluble factors released from fibroblasts did not affect collagen degradation by neutrophils. neutrophils are activated by type I collagen.(3) Expression of MMP-1 and MMP-3 in fibroblasts induced by neutrophil-CM. We examined the effects of neutrophil-CM on MMP expression by fibroblasts with the use of immunoblot analysis. Neutrophil-CM derived from collagen gel cultures did not contain proteins recognized by antibodies to rabbit pro-MMP-1, MMP-1, proMMP-3 and MMP-3. In contrast, medium obtained after culture of corneal fibroblasts in collagen gels produced a small amount of pro-MMP-1, active MMP-1, proMMP-3 and active MMP-3. The further addition of neutrophil-CM to the fibroblast cultures increased the abundance of both proMMP-1, active MMP-1and proMMP-3 and active MMP-3. We then investigated the effects of neutrophil-CM on the abundance of MMP mRNAs in corneal fibroblasts. the amounts of MMP-1 and MMP-3 mRNAs in the fibroblasts were determined by RT and real-time PCR analysis. Corneal fibroblasts contained both MMP-1 and MMP-3 mRNAs in the absence of neutrophil-CM. However, the amounts of both of these mRNAs were increased markedly by incubation of fibroblasts with neutrophil-CM.(4) To examine further whether the enhancement by neutrophils of collagen degradation by corneal fibroblasts is mediated by the up-regulation of MMP expression in fibroblasts, we investigated the effect of ilomastat, a synthetic inhibitor of MMPs on collagen degradation. The addition of ilomastat resulted in significant inhibition of the stimulatory effect of neutrophil-CM on collagen degradation by fibroblasts.(5) Effect of IL-1ra on collagen degradation by cocultures of neutrophils and corneal fibroblasts. IL-1ra inhibited collagen degradation in cocultures of neutrophils and corneal fibroblasts in a dose-dependent manner; IL-1ra had no effect on collagen degradation in cell-free cultures or in cultures of corneal fibroblasts or neutrophils alone. It did, however, inhibit in a dose-dependent manner collagen degradation by corneal fibroblasts in the presence of neutrophil-CM. These results suggest that IL-1 participates in the interaction between neutrophils and corneal fibroblasts that is responsible for stimulation of collagen degradation. (6) Inhibitory effect of Dexamethasone on collagen degradation by rabbit corneal fibroblasts. Dexamethasone inhibited in a dose-dependent manner the collagen degradation by corneal fibroblasts apparent in the absence or presence of neutrophil-CM. To investigate the mechanism by which dexamethasone inhibits collagen degradation by corneal fibroblasts. We examined its effects on the expression of MMPs in these cells by immunoblot analysis and gelatin zymography. Immunoblot analysis with antibodies to MMP-1 and MMP-3 revealed that Dexamethasone induced a dose-dependent decrease in the abundance of proMMP-1 and proMMP-3i in culture supernatans of corneal fibroblasts;Reverse transcription and real-time PCR showed that Dexamethasone had no significant effect on the basal abundance of MMP-1mRNA but significantly inhibited the effect of Neu-CM induced MMP-1mRNA and MMP-3mRNA. Gelatin zymography of culture supernatants showed that Dexamethasone reduced the amount of proMMP-9 in the absence or presence of Neu-CM, but it had no effect the amount of pro-MMP-2 or active MMP-2.Conclusions(1) Our results have shown that neutrophils stimulate collagen degradation by corneal fibroblasts, and that this effect is mediated through an increase in the expression of MMPs by the fibroblasts. This stimulatory effect of neutrophils was mimicked by neutrophil-CM and was much greater with CM derived from collagen gel cultures than with that prepared from monolayer cultures in the absence of collagen.(2) neutrophils stimulate collagen degradation by corneal fibroblasts not through cell-cell contact but rather through the release of a soluble factor, and that the release of such a factor by neutrophils is promoted by extracellular collagen. The soluble factor concerned with IL-1 because IL-1ra ingibited collagen degradation in cocultures of neutrophils and corneal fibroblasts in a dose-dependent manner. These results suggest that IL-1 participates in the interaction between neutrophils and corneal fibroblasts that is responsible for stimulation of collagen degradation.(3) Our results therefore suggest that neutrophils might act as regulators, rather than as effectors, of the degradation of extracellular collagen associated with corneal ulceration. corneal fibroblasts are the primary mediators of collagen degradation during corneal ulceration, and that infiltrated neutrophils serve to stimulate the collagenolytic activity of these resident cells of the corneal stroma.(4) Dexamethasone inhibed collagen degradation by corneal fibroblasts and Both the synthesis and activation of MMPs were inhibited by its. Suggest that corticosteroids therapeutics might prove effective for the treatment of some corneal ulceration .