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.

Increased interleukin-1 activity in the injured vitamin A-deficient cornea.

Injury to a vitamin A-deficient cornea leads to severe acute inflammation often culminating in ulceration. We report on possible regulatory mechanisms involved in the pathogenesis of corneal inflammation in vitamin A deficiency. Thymocyte comitogenic assay and interleukin (IL)-6 induction in corneal fibroblasts have shown that thermally injured and mechanically abraded vitamin A-deficient rat corneas produce much higher levels of an IL-1-like factor as compared with uninjured or injured, normal control corneas. This was confirmed by antibody capture enzyme immunoassay, which detected high levels of IL-1 alpha and IL-1 beta in injured vitamin A-deficient corneas. To our knowledge this is the first report describing the induction of IL-1 in the vitamin A-deficient cornea by thermal and mechanical injuries. When mechanically injured corneas were screened for chemotactic activity, they were found to contain significantly higher levels of a chemoattractant as compared with similarly injured, normal control corneas. Chemotactic activity [expressed as a percentage of a known chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine (fMLP), found in medium harvested from vitamin A-deficient corneas] averaged 58.8 +/- 8.9% (SEM) as compared with 12.6 +/- 5.4% in medium conditioned by normal corneas. Checkerboard analysis confirmed that the activity in vitamin A-deficient cornea conditioned medium was chemotactic and not chemokinetic. These results demonstrate a correlation between IL-1 levels and severity of inflammation in the injured vitamin A-deficient rat cornea.

Increased gelatinolytic and caseinolytic activity in the thermally injured, nutritionally compromised rat cornea: detection of a 27-kDa lymphoreticular cell-associated caseinase.

This study assesses the impact of various forms of injury on matrix degrading enzymes in nutritionally compromised rat corneas. In vitamin A-deficient (nutritionally compromised) and normal control corneas, in vivo or ex vivo mild mechanical abrasion did not appreciably alter the activity of either the 65-kDa or the 92-kDa gelatinases. In contrast, after thermal injury, while no appreciable change was detected in activity associated with the 65-kDa gelatinase in either vitamin A-deficient or normal control corneas, 92-kDa gelatinolytic activity was consistently higher in corneas from both groups, although activity associated with nutritionally compromised corneas was much higher. In these corneas, thermal injury also induced the expression of two high molecular weight (approximately 130-kDa and 225-kDa) gelatinases and a 27-kDa caseinase. While gelatinases were totally inactivated by inhibitors of metalloproteinases such as 1,10-phenanthroline and Galardin MPI, the 27-kDa caseinase showed considerable susceptibility to a mixture of serine protease inhibitors (aprotinin, dichloro-isocoumarin and pA-PMSF [(4-amidino-phenyl)-methane-sulphonyl fluoride]. Furthermore, unactivated-lymphoreticular cells from either nutritionally compromised or normal control animals contained a 24- and 27-kDa caseinase, however most of the activity was due to the 24-kDa caseinase. In contrast, glycogen-activated lymphoreticular cells contained a preponderance of the 27-kDa caseinase. Activated-lymphoreticular cells also expressed 92-kDa, 130-kDa and 225-kDa gelatinases. The presence of low molecular weight caseinases in lymphoreticular cells implicates them as the source of these enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 regulates the proliferation of leukocytes in human corneal cell-peripheral blood leukocyte cocultures.

We have documented the inability of human corneal epithelial-like cells to suppress proliferation of peripheral blood leukocytes (PBLs) induced by allogeneic PBLs in a mixed leukocyte reaction (MLR). Instead, enhanced proliferation of PBLs, albeit small, was consistently noted as indicated by uptake of radiolabeled thymidine. Maximum proliferation of PBLs was detected when a mixed leukocyte reaction (MLR) was conducted in the presence of corneal cells. High levels of interleukin-1 beta (IL-1 beta) were found during MLR irrespective of the presence of corneal cells. High levels of IL-1 beta correlated well with observed synergistic stimulation of PBL proliferation by corneal cells and stimulating allogeneic PBLs. In PBL-corneal cell cocultures, PBLs produced IL-1 beta; corneal cells contributed large amounts of prostaglandin E2 (PGE2). Although indomethacin completely blocked prostaglandin E2 production, it did not significantly alter the results. Our data show that PBLs and corneal cells can reciprocate each other's presence, and, under appropriate conditions, corneal cells can deliver at least one signal to enhance rather than suppress antigen-driven PBL proliferation. Our data suggest a role for immunoregulatory cytokines and prostanoids such as IL-1 beta and PGE2 in these interactions.

Effect of vitamin A deficiency on the adhesion of rat corneal epithelium and the basement membrane complex.

PURPOSE: To understand the underlying mechanisms responsible for the easy removal and sloughing of corneal epithelium in vitamin A deficiency. METHODS: An animal model of vitamin A deficiency, the vitamin A-deficient rat (A-rat), transmission electron microscopy, computer-assisted morphometric analysis and indirect immunofluorescence were used to study the adhesion of rat corneal epithelium to its basement membrane with emphasis on structure and molecular composition of the anchoring structures such as the hemidesmosome and bullous pemphigoid antigen. RESULTS: Transmission electron microscopy resolved numerous microseparations of the basal epithelial cell membrane from the basement membrane with intervening segmental basement membrane duplications and electron dense deposits. Morphometric analysis disclosed a statistically significant reduction in the frequency and size of hemidesmosomes. Four weeks after supplementing the diet with retinyl acetate (700 micrograms/week), significant reversal of these same structural abnormalities could be detected. Immunofluorescence staining for bullous pemphigoid antigen, a component of the adhesion complex, showed intense staining of the basal epithelial cytoplasm but weak and discontinuous staining of the basement membrane. Weak staining for laminin was also evident in A- corneas. In contrast, normal corneas displayed no cytoplasmic staining for bullous pemphigoid antigen and intense staining of the basement membrane for bullous pemphigoid antigen and laminin. CONCLUSIONS: The authors propose that structural abnormalities of the epithelial basement membrane complex are responsible for the observed loose epithelial adhesion and sloughing, as well as other known abnormalities of healing in the vitamin A-deficient rat cornea.

Cleavage and activation of corneal matrix metalloproteases by Pseudomonas aeruginosa proteases.

PURPOSE: To examine the effect of Pseudomonas aeruginosa on the expression of corneal matrix metalloproteases and the effect of its proteases on activation of corneal matrix metalloproteases in vitro. METHODS: Rat corneas and human corneal fibroblasts were co-cultivated with two different strains (RPS & 599A) of P. aeruginosa and one strain of Staphylococcus aureus, and the conditioned media were analyzed for proteolytic activity by gelatin and casein zymography. Human corneal fibroblast-conditioned medium was incubated with that from either strain of P. aeruginosa and was analyzed in a similar manner. RESULTS: Normal rat corneas in organ culture produce a 65 kDa gelatinase (inactive matrix metalloprotease-2), whereas thermally injured rat corneas additionally produce gelatinases with molecular masses of 92 kDa (inactive matrix metalloproteases-9) and > 200 kDa. Matrix metalloprotease-2 is also detected in human corneal fibroblast-conditioned medium. Although these matrix metalloproteases are no longer detectable when rat corneas or human corneal fibroblasts are co-cultured with two strains of P. aeruginosa for 48 hr, a 58 kDa gelatinase fragment appears in earlier stages of co-culture. In contrast, S. aureus does not affect matrix metalloprotease-2. The 58 kDa fragment is also evident by incubating human corneal fibroblast-conditioned medium with that from either strain of P. aeruginosa. Conditioned medium from the RPS strain, which produces both elastase and alkaline protease, is more effective in cleaving matrix metalloprotease-2 than that from the 599A strain, which expresses mainly alkaline protease. CONCLUSION: The secreted inactive corneal matrix metalloprotease-2 is activated through limited proteolysis by pseudomonal proteases.

Regulation of mitogen-driven lymphoreticular cell activation by human corneal cells and interleukin-1.

Keratin-positive fibroblast-like epithelial cells (FLE), isolated from human corneo-scleral-conjunctival rims, were shown to inhibit mitogen-driven (concanavalin A) DNA synthesis by murine thymocytes and splenocytes [lymphoreticular cells (LRC)]. The effect exerted by live cells in culture and by their supernatants was caused by factors active across species barriers. Paraformaldehyde-fixed or irradiated cells also suppressed mitogen-induced thymocyte DNA synthesis, but their supernatants manifested no such activity. Interaction between FLE cells and LRC in the presence of the mitogen resulted in suppressed cellular activation as evidenced by significantly lowered tetrazolium salt (MTT) reduction in murine thymocytes and splenocytes, suggesting reduced mitochondrial activity. The suppressive effect was seen with live and paraformaldehyde-fixed FLE cells. There was a good correlation between MTT assays and [3H]thymidine uptake experiments. Suppression of MTT reduction in murine thymocytes and splenocytes by intact FLE cells could be reversed by the addition of interleukin-1 (IL-1). Indomethacin prevented FLE-conditioned medium-induced suppression but failed to relieve suppression by whole FLE cells. Thus, suppression of LRC function by FLE cells and their secretions appeared to operate by different mechanisms. One mechanism related to prostaglandins present in FLE cell-conditioned medium, whereas another mechanism appeared to involve cell-membrane-associated factor(s). The findings not only provide additional information on the capability of corneal cells to regulate lymphoreticular cells but suggest an important role for IL-1 in the regulation of LRC function and corneal inflammation and immunity.

Proteolytic activation of corneal matrix metalloproteinase by Pseudomonas aeruginosa elastase.

Purified Pseudomonas aeruginosa elastase cleaved a 65 kDa gelatinase [inactive proenzyme form of matrix metalloproteinase (MMP-2)] from human corneal fibroblasts into a biologically active fragment with an approximate molecular mass of 58 kDa. However, purified pseudomonal alkaline protease did not cleave MMP-2 appreciably. Since activated MMP-2 is known to degrade native type IV, V and VII collagens, all components of the corneal basement membrane or stroma, our results suggest a new role for pseudomonal elastase in the pathogenesis of corneal infection, inflammation and ulceration.

Interferon-gamma, Staphylococcus aureus, and lipopolysaccharide/silica enhance interleukin-1 beta production by human corneal cells.

Cultures derived from human corneo-scleral rims remaining after a central corneal button had been removed for transplantation, revealed two types of cells on light microscopy: One with typical epithelial morphology and the other resembling fibroblasts. Both cell types contained keratin filaments in early passage and were therefore considered epithelial in nature. The fibroblast-like cells were designated fibroblast-like epithelial cells (FLE) while the typical epithelial cells were referred to as E-type. Both E and FLE cells constitutively produced an IL-1-like factor as determined by thymocyte proliferation assay and IL-2 induction in EL-4 lymphoma cells. Moreover, the supernatants from these cells potentiated concanavalin A (Con A)-primed mitochondrial oxidative metabolism in thymocytes, as indicated by the tetrazolium salt reduction assay (MTT) and this effect was significantly neutralized with monoclonal anti-IL-1 beta. The release of biologically active IL-1 beta by the FLE cells is another characteristic (in addition to the presence of keratin) distinguishing these cells from fibroblasts which do not release biologically active IL-1 beta. Using an ELISA, specific for IL-1 beta, there was clear cut evidence for increased production of this cytokine by FLE cells in response to human recombinant gamma-interferon (IFN-gamma), Staphylococcus aureus, and lipopolysaccharide (LPS) in combination with silica (LPS/silica). Time studies with IFN-gamma and LPS/silica demonstrated that enhanced production was time dependent and that IL-1 beta was primarily cell associated. The results indicate that human corneal E- and FLE-type cells can produce and release IL-1 and that FLE cells can be induced by inflammatory mediators to increase production of IL-1 beta.

Corneal epithelial cells produce thromboxane in response to interleukin 1 (IL-1).

Synthesis of thromboxane, a product of arachidonic acid formed via the cyclooxygenase pathway, was studied in rabbit corneal epithelial cells (SIRC cell line) under resting conditions and under the influence of interleukin 1 (IL-1). IL-1 potentiated the production of thromboxane 3-10-fold in a dose-dependent manner. This finding assumes added significance in view of the previous observations that the same cells are capable of producing IL-1. Thus, the corneal epithelial cells may be viewed in this context as an autocrine cell producing two biologically active substances which can serve as mediators of inflammation, one of which can augment the production of the other.