Role of serum amyloid A as an intermediate in the IL-1 and PMA-stimulated signaling pathways regulating expression of rabbit fibroblast collagenase.

The matrix metalloproteinase collagenase is expressed by resident tissue cells only when needed for biological remodeling. Exogenous addition of inflammatory and growth-promoting cytokines stimulates collagenase expression in early passage fibroblast cultures. In addition, the signal for collagenase expression in response to phorbol-12 myristate-13 acetate (PMA) or to agents which alter cell shape in early passage fibroblast cultures is routed extracellularly to an autocrine cytokine intermediate, IL-1 alpha. Importantly, fibroblasts, when freshly isolated from the tissue, are not competent for IL-1 alpha gene expression and, therefore, cannot produce collagenase in response to shape change agents. However, they do make a small amount of collagenase in response to PMA via an IL-1-independent pathway that has not been further characterized. In this paper, we investigate the role of a second autocrine, serum amyloid A3 (SAA3), in IL-1-dependent and -independent collagenase gene expression. We demonstrate that SAA3 is required for effective stimulation of collagenase expression by either exogenous or endogenous IL-1. Furthermore, while freshly isolated fibroblasts cannot express IL-1 alpha they can express SAA3, and this autocrine mediator acts independently of IL-1 alpha to control the low level of collagenase expression that can be stimulated by PMA. These results provide further evidence for a newly emerging paradigm of collagenase regulation which emphasizes the requirement for extracellular routing of signals. They also suggest that SAA3 might be utilized independently of IL-1 alpha to control tissue remodeling in vivo.

Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury.

Delayed re-epithelialization of the cornea after injury usually precedes stromal ulceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adhesive structures at the basement membrane zone. In this study, we provide additional evidence for this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibitions of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermally injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both could participate in dissolution of this structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types of corneal injury.

Unique regulation of the matrix metalloproteinase, gelatinase B.

PURPOSE: The matrix metalloproteinase (MMP), gelatinase B, is expressed by both corneal cell types found at the epithelial-stromal tissue interface, the site of basement membrane repair in the healing cornea. This study investigates the relative regulation of gelatinase B compared to other MMPs in response to agents related to those found in the corneal repair environment or in corneal ulcers. METHODS: A culture model of corneal cells isolated from rabbit was used. RESULTS: Gelatinase B is the major MMP expressed by corneal epithelial cells, whereas stromal fibroblasts produce gelatinase B along with three other MMPs: collagenase, stromelysin, and gelatinase A. Phorbol-12-myristate 13-acetate (PMA) stimulates gelatinase B mRNA and protein synthesis by corneal cells, which is similar to its effect on the other MMPs. Stimulation occurs, at least partially, at the transcriptional level. PMA-stimulated MMP expression follows biphasic kinetics, with the major effect on collagenase, stromelysin, and gelatinase A occurring during the late component. In contrast, the major gelatinase B response occurs during the early component. Transforming growth factor-beta (TGF-beta) has no effect on constitutive expression of gelatinase B by fibroblasts; however, expression stimulated by PMA is enhanced. In contrast, constitutive expression of collagenase and stromelysin is inhibited by TGF-beta. However, in the presence of PMA, the initial inhibitory effect of TGF-beta is reversed after treatment. CONCLUSION: Gelatinase B expression is regulated differently from other corneal MMPs. This provides a mechanism for control of basement membrane repair independent of repair processes in the stroma.

The rabbit gene for 92-kDa matrix metalloproteinase. Role of AP1 and AP2 in cell type-specific transcription.

We isolated the rabbit gene for the 92-kDa matrix metalloproteinase, gelatinase B, and sequenced 1802 contiguous bases covering the first three exons and 522 bases of DNA upstream of the start site for transcription. The DNA between bases -519 and +19 is sufficient to drive expression of a reporter gene in early passage cultures of corneal fibroblasts or primary cultures of corneal epithelial cells. Basal activity of the gelatinase B promoter in fibroblasts is lower than a collagenase promotor of 1800 base pairs, but activity of both promotors is similarly stimulated by treatment of transfected cells with phorbol 12-myristate 13-acetate, and stimulation is enhanced by co-treatment with transforming growth factor-beta. In contrast, basal activity of the gelatinase B promotor in epithelial cells is higher than the collagenase promotor. Deletion analysis demonstrated that sequences upstream of base -330 confer cell type-specific activity to the gelatinase B promotor. Site-directed mutagenesis revealed that an AP1-like element within this region is specifically utilized by fibroblasts. This region also contains elements that confer the capacity for activation by AP2, a transcription factor found to be expressed by corneal epithelial cells but not by corneal fibroblasts. In contrast, AP2 does not activate the collagenase promotor. These results provide a molecular basis for the unique cell type-specific expression pattern of gelatinase B as compared to other matrix metalloproteinases.

Interleukin 1 alpha mediates collagenase synthesis stimulated by phorbol 12-myristate 13-acetate.

Stimulation of collagenase expression in cultures of normal diploid fibroblasts by the tumor promotor phorbol 12-myristate 13-acetate (PMA) occurs secondarily to synthesis of unknown intermediary proteins. We have investigated the hypothesis that a form of the cytokine interleukin 1 (IL-1) is one intermediate controlling PMA-stimulated collagenase expression. Treatment with an IL-1 receptor antagonist inhibits the constitutive synthesis of collagenase in early passage fibroblast cultures from rabbit. Radioimmunoassay demonstrates that, of the two known IL-1 forms, IL-1 alpha and IL-1 beta, only IL-1 alpha is synthesized and released into the medium of corneal fibroblast cultures. PMA treatment of cells increases the level of IL-1 alpha mRNA; this occurs prior to the increase in collagenase mRNA and corresponds with increased synthesis and release of IL-1 alpha protein. Neutralizing antiserum to IL-1 alpha inhibits constitutive collagenase synthesis. Reagents that inhibit the activity of IL-1 alpha (IL-1 receptor antagonist or neutralizing antibody) also inhibit the PMA-mediated stimulation of collagenase synthesis. These results indicate that constitutive and PMA-stimulated expression of collagenase is regulated through an IL-1 alpha intermediate. In vivo, regulation of the lytic phase of tissue remodeling through the IL-1 alpha intermediate may ensure the recruitment of cells adjacent to the one that received the initial stimulus.

Stromal fibroblasts synthesize collagenase and stromelysin during long-term tissue remodeling.

The process of connective tissue remodeling is an important mechanism contributing to tissue morphogenesis in development and homeostasis. Although it has long been known that remodeling tissues actively mediate collagenolysis, little is understood about the molecular mechanisms controlling this cell-regulated process. In this study, we examined the biosynthesis of collagenase and the related metalloproteinase, stromelysin, during remodeling of repair tissue deposited after mechanical injury to the rabbit cornea. Neither enzyme was synthesized by uninjured corneas; however, synthesis and secretion was detectable within one day after injury. Collagenase accumulated in its latent form while stromelysin appeared to be partially activated. Enzymes were synthesized by cells having a fibroblast phenotype. These cells were found within the stroma. New synthesis was correlated with accumulation of enzyme-specific mRNA. Highest levels of enzyme synthesis were observed in the repair tissue. However, stromal cells outside of the repairing area also synthesized both enzymes. The level of synthesis decreased in a gradient radiating from the repair tissue. Total synthetic levels in a given area of cornea were dependent on both the number of cells expressing enzyme and the rate of enzyme synthesis. Synthesis of collagenase was detected in repair tissue as long as nine months after injury. Our findings provide direct support for the hypothesis that new collagenase synthesis by cells in repair tissue is the first step in collagen degradation during long-term tissue remodeling.

Collagenolytic/gelatinolytic enzymes in corneal wound healing.

We have documented changes in expression of collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in healing or ulcerating corneal wounds of rat or rabbit. Correlation of our findings with specific changes in the extracellular matrix of the repair tissue suggests two different roles for the enzymes, MMP-2 and MMP-9. MMP-2 is expressed in undamaged corneal stroma where it may degrade the occasional collagen molecule that becomes damaged. After corneal wounding, expression of this enzyme is increased and much of it appears in the active form. These changes persist for at least seven months, suggesting that MMP-2 is involved in the prolonged process of collagen remodelling in the stromal repair tissue. MMP-9 is expressed in the epithelial layer of repair tissue with a timing suggesting it might participate in controlling resynthesis of the basement membrane. MMP-9 also appears to be involved in degradation of the epithelial basement membrane that precedes corneal ulceration.

Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea.

We have documented changes in collagenolytic/gelatinolytic enzymes of the matrix metalloproteinase family (MMP) in remodelling rabbit cornea. MMP-2 (65 kDa gelatinase) in the proenzyme form is synthesized by the cells of the normal corneal stroma. After keratectomy the level of MMP-2 is increased in the stroma and enzyme appears in both pro- and activated forms. In addition, corneal cells synthesize MMP-9 (92 kDa gelatinase) in the proenzyme form after keratectomy; expression occurs in both the epithelial as well as stromal corneal layers. Changes in expression of both enzymes are precisely localized to the repairing portion of cornea, but demonstrate important differences in timing that correlate with the timing of specific events of matrix remodelling. Our data suggest that each of the gelatinases plays a different role in tissue remodelling after injury. We hypothesize that MMP-2 performs a surveillance function in normal cornea, catalyzing degradation of collagen molecules that occasionally become damaged. After wounding, this enzyme appears to participate in the prolonged process of collagen remodelling in the corneal stroma that eventually results in functional regeneration of the tissue. MMP-9 expression does not correlate with stromal remodelling, but we suggest that the enzyme might play a part in controlling resynthesis of the epithelial basement membrane.

Transforming growth factor-beta and interleukin-1 modulate metalloproteinase expression by corneal stromal cells.

The enzyme collagenase participates in remodeling the extracellular matrix of corneal stroma during normal wound healing and mediates the degradation of extracellular matrix that occurs in many corneal pathologic states. Because this enzyme is synthesized and secreted by corneal cells, therapy of degradative disorders might be geared toward control of enzyme expression. The effects of two cytokines, transforming growth factor-beta (TGF-beta) and interleukin-1 (IL-1), on the expression of collagenase by cultured corneal stromal cells are reported. In addition, the concomitant effects of these cytokines on expression of three additional members of the matrix metalloproteinase (MMP) family--stromelysin, 72-kilodalton (kD) gelatinase, and 92-kD gelatinase--were investigated. When stromal cells are situated in the normal corneal stroma, they produce only a single MMP, 72-kD gelatinase. This pattern of expression was reproduced by stromal cells freshly plated in primary culture. However after passage in culture, the cells also began to secrete collagenase and stromelysin. Treatment of primary cultures with recombinant human IL-1 also induced collagenase and stromelysin expression. In addition, 92-kD gelatinase expression was induced and 72-kD gelatinase expression was increased further by IL-1 treatment. Treatment of passaged cultures or IL-1-treated primary cultures with recombinant human TGF-beta reverted the pattern of enzyme expression toward that exhibited by primary, untreated cultures, ie, expression of collagenase and stromelysin was repressed while expression of 72-kD gelatinase was increased. These results suggest that TGF-beta and IL-1 may be important agents for controlling MMP expression in healthy and diseased corneas.

The pattern of metalloproteinase expression by corneal fibroblasts is altered by passage in cell culture.

We have examined the pattern of expression of four different matrix metalloproteinases (MMPs), collagenase, stromelysin, 92 kD gelatinase, and 72 kD gelatinase, by primary and passaged cultures of rabbit corneal fibroblasts. Primary cultures of this cell type have previously been shown to reproduce the normal tissue regulation of collagenase expression. We demonstrate qualitative and quantitative changes in the pattern of MMP expression as the cells are passaged in culture. Only a single MMP, 72 kD gelatinase, is constitutively expressed by primary fibroblast cultures. Phorbol myristate acetate (PMA) treatment upregulates expression of 72 kD gelatinase and turns on the expression of collagenase and stromelysin, as well as 92 kD gelatinase. However, the degree to which MMP expression is induced is minimal. Cells subcultured but a single time constitutively produce not only 72 kD gelatinase, but also collagenase and stromelysin. In addition, PMA treatment upregulates expression of collagenase, stromelysin and 92 kD gelatinase to high levels. In contrast, the expression of 72 kD gelatinase is repressed by treatment of passaged cell cultures with PMA. Our data indicate that the cell does not simply turn the MMP genes on or off, as a group, in response to various agents, but that it has the capacity for fine control over which MMPs are expressed and the degree to which each is expressed. Changes in MMP protein expression induced by PMA treatment are correlated with changes in specific mRNA levels in passaged cultures. The kinetics of mRNA accumulation suggest that the MMP genes can respond to multiple intracellular signals initiated in a temporal cascade by PMA. It is the combined effects of the individual signals on the accumulation of specific mRNAs that must determine the ultimate pattern of MMP protein expression. The distinct patterns of MMP expression produced by primary and passaged cell cultures may be analogous to patterns of expression that might occur under particular in vivo conditions.

Expression of collagenolytic/gelatinolytic metalloproteinases by normal cornea.

Members of the gelatinase subclass of the matrix metalloproteinase family have the capacity to degrade denatured collagens of all types and native types IV, V, and VII collagens. The authors identified the metalloproteinase species of the gelatinase class produced by the cells of rabbit corneal tissue. Two different molecular forms of gelatinase, visualized as enzymatic activities, that undergo electrophoresis with different mobilities on gelatin zymograms are synthesized by corneal cells in serum-free organ culture. The enzyme species that has the slower mobility is biochemically and immunologically related to a gelatinase synthesized by macrophages and neutrophils which has been called both type IV and type V collagenase. The second gelatinase species is related to a second enzyme, the product of a different gene, which has also been called type IV collagenase. The electrophoretic mobilities of these enzymes on polyacrylamide gels indicate the inactive proenzyme forms. The authors refer to these enzymes as 92-kilodalton (kD) gelatinase and 72-kD gelatinase based on their electrophoretic mobilities under sulfhydryl-reducing conditions. In primary cell culture, corneal epithelial cells were found to synthesize predominantly the 92-kD gelatinase species whereas the 72-kD gelatinase is synthesized mostly by stromal fibroblasts. However, each cell type can produce small amounts of the other enzyme. The 72-kD gelatinase, mostly in the proenzyme form, can be extracted from the normal corneal stroma without culturing, but expression of 92-kD gelatinase can only be detected in cell or organ culture. The substrate specificities of these enzymes suggests that they may be of central importance in the degradation of the epithelial basement membrane and in formation of the epithelial defect that precedes corneal ulceration.

Glucocorticoid effects on the production and actions of immune cytokines.

The immunosuppressive actions of glucocorticoids form the basis for their use in treatment of autoimmune diseases and prevention of allograft rejection. However, the mechanisms responsible for glucocorticoid-induced immunosuppression are still poorly understood. It is now clear that glucocorticoids do not inhibit all aspects of the immune response and, in some cases, may enhance certain functions of immune effector cells. One example is that of the dramatic increase induced by IFN-gamma in the number of IgG Fc receptors on human mononuclear phagocytes, which is enhanced rather than inhibited by glucocorticoids. An aspect of the immune response which appears to be consistently suppressed by glucocorticoids is the production of immune cytokines. Since these hormones appear to be essential mediators for a vigorous immune response, inhibition of their production may be an effective way for glucocorticoids to block the immune response.

Glucocorticoids enhance the gamma-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression.

At physiologic and therapeutic concentrations, glucocorticoids decrease the number of Fc receptors for IgG (Fc gamma R) on human monocyte-like cell lines. In comparison, gamma-interferon (IFN-gamma) increases Fc gamma R expression on both human monocytes and monocyte-like cell lines. In this study, we examined the combined effects of glucocorticoids and IFN-gamma on human monocyte expression of the high affinity (72 kDa) Fc gamma R. Mononuclear cells prepared from heparinized venous blood of normal donors were treated for up to 90 hr with or without recombinant IFN-gamma and/or steroids. Monocyte Fc gamma R were measured by Scatchard analysis of the binding of human monomeric 125I-IgG1; indirect immunofluorescence plus flow cytometry, utilizing a monoclonal antibody (MoAb 32) which is specific for the high affinity Fc gamma R; and direct immunofluorescence using fluorescein isothiocyanate-labeled human monomeric IgG1 and flow cytometry quantitated using U-937 cells as a standard. Cultured monocytes incubated in the presence of both glucocorticoids and IFN-gamma for 18 hr had significantly higher (p less than 0.01) Fc gamma R levels than monocytes treated with IFN-gamma alone. The effect of combined treatment reached a plateau by 42 hr of incubation without increasing expression of other surface markers tested. Treatment with glucocorticoids alone did not consistently decrease monocyte Fc gamma R levels after either 18 or 42 hr of culture. Only glucocorticoids augmented the IFN-gamma increase in Fc gamma R; other steroids tested had no effect on IFN-gamma action. Furthermore, the effect was observed after treatment with only one type of interferon, IFN-gamma. These results describe a glucocorticoid immunoregulatory effect that may explain why combined IFN-gamma plus glucocorticoid treatment enhances mononuclear phagocyte Fc-mediated functions.