Molecular identification of t4 and t5 genotypes in isolates from acanthamoeba keratitis patients.

Acanthamoeba keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the Rns genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.

Matrix metalloproteinase 2 gene knockout has no effect on acute brain injury after focal ischemia.

Matrix metalloproteinases (MMPs) may contribute to tissue damage after cerebral ischemia. In this study, wildtype and MMP-2 knockout mice were subjected to permanent and transient (2 h) occlusions of the middle cerebral artery. Gelatin zymography showed that MMP-9 levels were increased in all brains after ischemia. MMP-2 levels did not show a significant increase in wildtype mice, and were not detectable in knockout mice. Laser doppler flowmetry demonstrated equivalent ischemic reductions in perfusion in wildtype and knockout mice. In both permanent and transient occlusion paradigms, there were no statistically significant differences between wildtype and knockout mice in terms of 24 h ischemic lesion volumes. These data suggest that MMP-2 does not contribute to acute tissue damage in this model of focal ischemia.

Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia.

Deleterious processes of extracellular proteolysis may contribute to the progression of tissue damage after acute brain injury. We recently showed that matrix metalloproteinase-9 (MMP-9) knock-out mice were protected against ischemic and traumatic brain injury. In this study, we examined the mechanisms involved by focusing on relevant MMP-9 substrates in blood-brain barrier, matrix, and white matter. MMP-9 knock-out and wild-type mice were subjected to transient focal ischemia. MMP-9 levels increased after ischemia in wild-type brain, with expression primarily present in vascular endothelium. Western blots showed that the blood-brain barrier-associated protein and MMP-9 substrate zonae occludens-1 was degraded after ischemia, but this was reduced in knock-out mice. There were no detectable changes in another blood-brain barrier-associated protein, occludin. Correspondingly, blood-brain barrier disruption assessed via Evans Blue leakage was significantly attenuated in MMP-9 knock-out mice compared with wild types. In white matter, ischemic degradation of the MMP-9 substrate myelin basic protein was significantly reduced in knock-out mice compared with wild types, whereas there was no degradation of other myelin proteins that are not MMP substrates (proteolipid protein and DM20). There were no detectable changes in the ubiquitous structural protein actin or the extracellular matrix protein laminin. Finally, 24 hr lesion volumes were significantly reduced in knock-out mice compared with wild types. These data demonstrate that the protective effects of MMP-9 gene knock-out after transient focal ischemia may be mediated by reduced proteolytic degradation of critical blood-brain barrier and white matter components.

Induction of the ubiquitin-proteasome pathway during the keratocyte transition to the repair fibroblast phenotype.

PURPOSE: To examine dynamics and function of the ubiquitin (Ub)-proteasome pathway (UPP) during corneal stromal cell acquisition of the repair fibroblast phenotype. METHODS: An established cell culture model was used in which freshly isolated rabbit corneal stromal cells acquire a repair fibroblast phenotype, thereby mimicking injury-induced stromal cell activation. RESULTS: Transition to the repair fibroblast phenotype during the 72 hours after initial plating was coincident with progressive UPP induction. Levels of Ub, Ub-conjugated proteins, ubiquitinylating enzymes E1 and E2-25K, and 26 S proteasome increased two- to fivefold in activated stromal cells. These increases were associated with enhanced (>10-fold) capacity for Ub-dependent proteolysis of (125)I-labeled H2A and with progressive (>6-fold) increases in the UPP substrate, inhibitor of kappaBalpha (IkappaBalpha). Because IkappaBalpha expression is induced by nuclear factor (NF)-kappaB, this finding suggests that rates of constitutive NF-kappaB activation, and thus IkappaBalpha degradation, are elevated in activated stromal cells. Both freshly isolated and activated stromal cells degraded IkappaBalpha in response to IL-1alpha; yet, only activated stromal cells maintained autocrine IL-1alpha expression after 24 hours. UPP induction was coincident with a more than 90% loss of tissue transketolase (TKT) and aldehyde dehydrogenase (ALDH) class 1. TKT was stabilized during the repair phenotype transition by proteasome inhibition and was degraded (>30%/h) by the UPP in cell-free assays. CONCLUSIONS: Coordinate induction of the UPP during stromal cell activation alters levels of IkappaBalpha and TKT, two UPP substrates that are implicated in the loss of tissue stasis and corneal clarity after injury.

Trefoil peptides promote restitution of wounded corneal epithelial cells.

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype.

The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-kappaB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye's aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.

Matrix metalloproteinase 9 promoter activity is induced coincident with invasion during tumor progression.

Matrix metalloproteinase 9 (MMP-9, also known as gelatinase B or 92-kd Type IV collagenase) is overexpressed in many human and murine cancers. We induced carcinomas in mice carrying a transgene that links the MMP-9 promoter to the reporter ss-galactosidase so that activation of the MMP-9 promoter would be indicated by ss-galactosidase. Mammary carcinomas were induced by mating the MMP-9 promoter reporter transgenic mice with mice carrying a transgene for murine mammary tumor virus promoter linked to polyoma middle T antigen, a transgene that leads to rapid development of mammary tumors in female mice. None of the hyperplastic mammary glands and none of the carcinomas in situ expressed ss-galactosidase. However, all invasive tumors had evidence of ss-galactosidase expression. In addition to the breast carcinomas, a malignant teratoma in a female and a papillary adenocarcinoma in the pelvic region of a male arose and were also ss-galactosidase positive. We also induced skin tumors in the mice with the MMP-9 reporter transgene with 7, 12-dimethylbenz[a]anthracene (DMBA) treatment followed by phorbol 12 myristate 13-acetate (TPA). None of the papillomas or in situ carcinomas showed any ss-galactosidase expression, but expression was seen in invasive carcinoma. Although normal skin epithelial cells did not express ss-galactosidase, we did find staining in a few cells at the duct of the sebaceous gland at the base of the hair follicles. The MMP-9 reporter transgene did not lead to expression in the alveolar macrophages, confirming that additional upstream sequences are required for expression in macrophages. These experiments have revealed that MMP-9 promoter activity is induced coincident with invasion during tumor progression. Furthermore, this indicates that the more proximal upstream elements of the promoter are sufficient for MMP-9 transcription during tumor progression.

Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94.

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches. Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size. However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury.

Matrix metalloproteinases (MMPs) belong to a class of extracellular proteinases responsible for maintaining and remodeling the extracellular matrix. In addition to multiple functions in normal physiology, abnormal MMP expression and activity may also participate in the pathophysiology of cerebral disease. Here, we show that MMP-9 (gelatinase B; EC.3.4.24.35) contributes to the pathophysiology of traumatic brain injury. After controlled cortical impact in mice, MMP-9 was increased in traumatized brain. Total MMP-9 levels at 24 hr were significantly increased as measured by a substrate cleavage assay. Zymograms showed that MMP-9 was elevated as early as 3 hr after traumatic brain injury, reaching a maximum at approximately 24 hr. Increased MMP-9 levels persisted for up to 1 week. Western blot analysis indicated increased profiles of MMP-9 expression that corresponded with the zymographic data. Knock-out mice deficient in MMP-9 gene expression were compared with wild-type littermates in terms of morphological and motor outcomes after trauma. Motor outcomes were measured at 1, 2, and 7 d after traumatic brain injury by the use of a rotarod device. MMP-9 knock-out mice had less motor deficits than wild-type mice. At 7 d, traumatic brain lesion volumes on Nissl-stained histological sections were significantly smaller in MMP-9 knock-out mice. These data demonstrate that MMP-9 contributes to the pathophysiology of traumatic brain injury and suggest that interruption of the MMP proteolytic cascade may be a possible therapeutic approach for preventing the secondary progression of damage after brain trauma.

Pax-6 expression and activity are induced in the reepithelializing cornea and control activity of the transcriptional promoter for matrix metalloproteinase gelatinase B.

Recent evidence supports the idea that matrix metalloproteinases (MMPs) act as morphogenetic regulators in embryonic and adult events of tissue remodeling. MMP activity is controlled primarily at the level of gene expression. In a recent study we characterized the transcriptional promoter of the MMP gene, gelatinase B (gelB), in transgenic mice, demonstrating the requirement for DNA sequences between -522 and +19 for appropriate activity. In this study we investigated factors required for gelB promoter activity in the developing eye and reepithelializing adult cornea. Pax-6 is a homeobox and paired domain transcription factor that acts at the top of the hierarchy of genes controlling eye development. Pax-6 is also expressed in the adult eye. We show here that the tissue expression pattern of Pax-6 overlaps extensively with gelB promoter activity in the developing and adult eye. In addition Pax-6 is observed to be upregulated in repairing corneal epithelium, as is gelB promoter activity. In cell culture transfection experiments, we identified two promoter regions which mediate positive response to Pax-6. By electrophoretic mobility shift assay, we further pinpoint two Pax-6 binding sites within these response regions and demonstrate direct interaction of the Pax-6 paired domain with one of these sites. These data suggest a mechanism by which Pax-6 may direct gelB expression in an eye-specific manner.

Curcuminoids inhibit the angiogenic response stimulated by fibroblast growth factor-2, including expression of matrix metalloproteinase gelatinase B.

We have studied mechanisms controlling activation of the gelatinase B gene (matrix metalloproteinase-9) by fibroblast growth factor-2 (FGF-2) during angiogenesis, and the effects of the natural product curcuminoids on this process. Using a transgenic mouse (line 3445) harboring a gelatinase B promoter/lacZ fusion gene, we demonstrate FGF-2 stimulation of reporter gene expression in endothelial cells of invading neocapillaries in the corneal micropocket assay. Using cultured corneal cells, we show that FGF-2 stimulates DNA binding activity of transcription factor AP-1 but not NF-kappaB and that AP-1 stimulation is inhibited by curcuminoids. We further show that induction of gelatinase B transcriptional promoter activity in response to FGF-2 is dependent on AP-1 but not NF-kappaB response elements and that promoter activity is also inhibited by curcuminoids. In rabbit corneas, the angiogenic response induced by implantation of an FGF-2 pellet is inhibited by the co-implantation of a curcuminoid pellet, and this correlates with inhibition of endogenous gelatinase B expression induced by FGF-2. Angiostatic efficacy in the cornea is also observed when curcuminoids are provided to mice in the diet. Our findings provide evidence that curcuminoids target the FGF-2 angiogenic signaling pathway and inhibit expression of gelatinase B in the angiogenic process.

Failure to activate transcription factor NF-kappaB in corneal stromal cells (keratocytes).

PURPOSE: Freshly isolated cultures of corneal stromal cells (keratocytes) are incompetent to synthesize the tissue remodeling proteinase, collagenase, in response to agents such as cytochalasin B (CB) or phorbol myristate acetate (PMA), which are strong stimulators of collagenase expression in subcultured fibroblasts of all types, including those from corneal stroma. Incompetence is due to failure to activate an autocrine interleukin (IL)1alpha feedback loop required to mediate cell response. The goal of the present study was to investigate the mechanism for this failure. METHODS: A cell culture model of freshly isolated corneal stromal cells and subcultured stromal fibroblasts from rabbits was used for these studies. RESULTS: Competence to synthesize collagenase in response to CB was acquired as a differentiation property by corneal stromal cells placed in culture, and did not require subculture. Competence acquisition correlated with transition to a fibroblastic spindle shape, assembly of actin stress fibers, and the acquired capacity to collapse in response to CB. It was demonstrated that competence could be more precisely defined as the capacity to express IL-1alpha in response to IL-1, making possible activation of the feedback loop. Investigation into the signaling pathway for IL-1alpha expression in response to IL-1 revealed a requirement for reactive oxygen species and activity of the transcription factor nuclear factor (NF)kappaB. Importantly, freshly isolated stromal cells were found to be relatively incompetent to activate NF-kappaB in comparison to subcultured stromal fibroblasts. CONCLUSIONS: Failure to activate NF-kappaB explains incompetence for expression of IL-1alpha in corneal stromal cells. Because NF-kappaB regulates many cell functions with potential to disturb corneal structure, including expression of inflammatory, stress, and degradative proteinase genes; protection against apoptosis; and cell replication; this seems likely to be an important mechanism protecting corneal stasis and preserving function.

Differential inhibition of collagenase and interleukin-1alpha gene expression in cultured corneal fibroblasts by TGF-beta, dexamethasone, and retinoic acid.

PURPOSE: Expression of the genes for collagenase and interleukin-1alpha (IL-1alpha) are induced as stromal cells become activated to the repair fibroblast phenotype after injury to the cornea. This investigation examines the mechanisms whereby expression of these genes is inhibited by transforming growth factor-beta (TGF-beta), dexamethasone (DEX), or retinoic acid (RET A). METHODS: A model of freshly isolated cultures of corneal stromal cells and early passage cultures of corneal fibroblasts was used in these studies. This model reproduces the events of stromal cell activation in the corneal wound. RESULTS: In early passage cultures of corneal fibroblasts, expression of collagenase is under obligatory control by autocrine IL-1alpha. IL-1alpha controls its own expression through an autocrine feedback loop that is dependent on transcription factor NF-kappaB. TGF-beta, DEX, and RET A were each effective inhibitors of collagenase gene expression in these cells. Furthermore, these agents have the capacity to inhibit expression of IL-1alpha and this was correlated with their ability to affect DNA-binding activity of NF-kappaB. However, TGF-beta, DEX, and RET A were also effective inhibitors of the low level of collagenase expressed by freshly isolated corneal stromal cells that cannot express IL-1alpha. CONCLUSIONS: In cells with an active IL-1alpha autocrine loop there are at least two distinct signaling pathways by which collagenase gene expression can be modulated. The results of this study demonstrate that TGF-beta, DEX, and RET A differentially inhibit collagenase and IL-1alpha gene expression. This information will be useful in the design of therapeutic modalities for fibrotic disease in the cornea and other parts of the eye.

Keratocyte and fibroblast phenotypes in the repairing cornea.

In mammals, tissue damage is usually repaired by activation of a fibrotic response which saves the life of the organism, but which can never restore function to the damaged organ. In addition, fibrotic responses form the basis for diverse pathologies, including many that occur in the eye. It is intriguing, therefore, to observe the occasional circumstances in which repair in mammals appears to take on a regenerative character, such as during fetal wound healing or in certain types of corneal wounds. The thesis of this chapter is that the choice between regeneration or fibrosis lies in the control of fibroblast phenotype. The cornea of the eye has several features which make it a particularly useful model for the study of fibroblast phenotype. Studies discussed herein, identify failure to activate the transcription factor NF-kappaB as a control mechanism for inhibiting fibroblast activation in the cornea. Evidence is further presented for the view that transition in fibroblast phenotype in repair tissue is not simply a matter of differential gene expression, but is a developmental event which reflects changes in the hard wiring of signalling pathways by which the cell responds to environmental input.

Regulation of gelatinase B production in corneal cells is independent of autocrine IL-1alpha.

PURPOSE: The matrix metalloproteinase gelatinase B is synthesized by cells at the leading edge of the corneal epithelium migrating to heal a wound. Recent data from the authors' laboratory suggest that excessive synthesis contributes to repair defects. The goal of the study reported here was to investigate mechanisms controlling gelatinase B production by corneal epithelial cells. METHODS: Freshly isolated cultures of corneal epithelial cells and early passage stromal fibroblasts from rabbit were used for these studies. RESULTS: In a previous study, it was found that the cytokine interleukin (IL)-1alpha is released into the culture medium of corneal epithelial cells more efficiently when they are plated at low density with limited cell-cell contact than when plated at high density. In this study, we show that production of gelatinase B by these cells is similarly affected by cell plating density. However, it is further demonstrated that these two events are not dependent on one another but occur in parallel: IL-1alpha does not regulate gelatinase B production (synthesis), nor was there evidence that any other secreted autocrine cytokine acts as mediator. Instead, our data suggest that gelatinase B production is downregulated directly by high cell density and indicate a connection to the level of protein kinase C activity. Nevertheless, the anticancer agent suramin, which blocks collagenase synthesis by interfering with autocrine cytokine-receptor interactions, still inhibits synthesis of gelatinase B. CONCLUSIONS: Unlike collagenase synthesis by corneal stromal fibroblasts, production (synthesis) of gelatinase B does not appear to be controlled by secreted autocrine cytokines but can still be inhibited by suramin. Suramin may make an effective therapeutic agent for controlling pathologic overproduction of gelatinase B in corneal ulcers.

Gelatinase B/lacZ transgenic mice, a model for mapping gelatinase B expression during developmental and injury-related tissue remodeling.

Matrix metalloproteinases (MMPs) drive normal tissue remodeling and are implicated in a wide range of pathologies. Although MMP activity is controlled at multiple levels, the primary regulation of MMP activity is transcriptional. The transcriptional promoter elements required for MMP gene expression in cultured cells have been defined, but this has not been extended to the in vivo situation. In this paper, we show that the DNA sequences between -522 and +19 of the rabbit gelatinase B gene (MMP-9) (as characterized in the transgenic mouse line 3445) constitute a minimal promoter that drives appropriate developmental and injury-induced reporter gene expression in transgenic mice. We further show that the expression and activity of three transcription factors (NF-kappaB, AP-2, and Sp1) that control the activity of the gelatinase B promoter are selectively induced in the epithelium migrating to heal a wound. Although promoter activity parallels expression of the endogenous gene in cell cultures, we show by several criteria that cell cultures cannot model many aspects of promoter regulation in vivo. This study reveals that the transgenic mouse line 3445 might be a useful model for investigating the regulation of gelatinase B expression in vivo and for identifying and characterizing new drugs that can control gelatinase B gene transcription.

Proteolytic mechanisms in corneal ulceration and repair.

Corneal stromal ulceration is a devastating disorder that can cause blindness. Stromal ulceration was once thought to be a physical dissolution process, which even now is described as "melting." However, a major paradigm change occurred about 25 years ago with the demonstration of extracellular matrix-degrading activity associated with tissues isolated from ulcerating corneas. Recent studies have identified the enzymes involved as specific members of the matrix metalloproteinase (MMP) family. These studies have further provided evidence that MMPs participate at all stages of the ulcerative process, from formation of the initiating epithelial defect to ulcer resolution and repair. Roles for MMPs in these processes are discussed in this review. Studies on corneal ulceration provide basic information about failure to heal, which is useful for understanding mechanisms common to other organ systems besides the cornea.

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Frog PNKT-4B cells express specific extracellular matrix-degrading enzymes and cytokines correlated with an invasive phenotype.

A temperature-dependent metastatic phenotype reported for a frog cell line, PNKT-4B, provided a means for studying potential mediators of cell-matrix interaction involved in metastatic invasion. Zymography revealed that these cells secreted enzyme species with properties and characteristics of mammalian metalloproteinases: collagenase, stromelysin, gelatinase A, and gelatinase B. These enzymes were produced by PNKT-4B cultures maintained at both invasive-permissive (28 degrees C), and invasion-restrictive (20 degrees C) temperatures. However, under the invasive-permissive culture condition cells produced more of the putative gelatinase B and A enzymes. In addition, an activated form of gelatinase A was produced only in invasion-permissive cultures. DNA synthesis bioassays (Mv1Lu cell line and mouse thymocytes) to detect growth promoting and/or inhibitory cytokines, revealed that PNKT-4B cultures kept at 28 degrees C released significantly higher levels of stimulatory (interleukin-1-like) and latent inhibitory (transforming growth factor-beta-like) substances into the medium compared to 20 degrees C cultures. Pre-absorption of media samples with heparin-sepharose indicated a second stimulatory cytokine as well. A corneal fibroblast bioassay that tests for mediators of collagenase synthesis, detected a stimulatory substance whose activity was greatly reduced in the presence of interleukin-1 receptor antagonist protein. Collagenase stimulatory activity present in 28 degrees C culture medium was significantly higher than equal samples from 20 degrees C cultures. These studies provide a molecular correlation between release of cytokines with properties of the metastatic phenotype seen in vivo. They further provide some of the first characterizations of frog MMPs and cytokines, which are likely to be involved in other tissue remodeling events.

Repair phenotype in corneal fibroblasts is controlled by an interleukin-1 alpha autocrine feedback loop.

PURPOSE: To explore the role of autocrine interleukin-1 alpha (IL-1 alpha) as a central regulator of the repair phenotype in corneal fibroblasts. METHODS: Disruption of the actin cytoskeleton with cytochalasin B (CB), which mimics changes in shape that occur in repair tissues, was used to stimulate repair gene expression in early-passage fibroblasts. Changes in expression of IL-1 alpha, IL-8, collagenase, and ENA-78 were determined by Northern blot analysis, radioimmunoassay, and an enzyme-amplified sensitivity immunoassay (EASIA). Expression of repair genes was also examined in repair fibroblasts, isolated from healing, penetrating keratectomy wounds in rabbits. RESULTS: Blocking IL-1 alpha activity prevented both constitutive and stimulated increases in synthesis of IL-8 and collagenase in early-passage cultures of corneal fibroblasts, demonstrating the role of IL-1 alpha as a necessary intermediate for expression of these genes. Evidence is also presented that the IL-1 alpha autocrine controls expression of an IL-8 related factor, ENA-78. Unlike early-passage fibroblasts, fibroblasts freshly isolated from the uninjured cornea did not express IL-1 alpha. However, fibroblasts freshly isolated from remodeling corneal repair tissue 3 weeks after injury were found to express substantial levels of IL-1 alpha, regulated through an autocrine feedback loop. Neutralization experiments demonstrated that the IL-1 alpha autocrine is largely responsible for controlling both collagenase and IL-8 synthesis in repair fibroblasts, as it is in early-passage fibroblasts. CONCLUSIONS: These findings provide evidence that activation of an autocrine IL-1 alpha feedback loop is an important mechanism by which fibroblasts adopt a repair phenotype during remodeling of the cornea.