Lactoferrin Glu561Asp facilitates secondary amyloidosis in the cornea.

AIM: To elucidate the pathogenic mechanism of amyloid formation in corneal amyloidosis with trichiasis. METHODS: Ophthalmological examination was performed in nine patients to determine secondary corneal amyloidosis with trichiasis. Congo red staining and immunohistochemistry using anti-human lactoferrin antibody were used for biopsied corneal samples. For genetic analyses, single strand conformation polymorphism (SSCP), direct DNA sequence analysis, and polymerase chain reaction (PCR) induced mutation restriction analysis (IMRA) were employed to detect lactoferrin gene polymorphism. RESULTS: All patients had had trichiasis at least for 1 year, and all amyloid-like deposits were found in one eye with trichiasis. Ophthalmological examination revealed that eight patients showed gelatinous type of amyloid deposition and one showed lattice type of amyloid deposition. Studies of biopsied corneal samples with Congo red stain revealed positive staining just under the corneal epithelial cells. Immunoreactivity of anti-human lactoferrin antibodies was recognised in all tissues with positive Congo red staining. Lactoferrin gene analysis revealed that seven patients were heterozygotic and two were homozygotic for lactoferrin Glu561Asp. The frequency of the polymorphism in the patients was significantly different from that in 56 healthy control subjects. CONCLUSION: Lactoferrin Glu561Asp is a key polymorphism related to facilitating amyloid formation in corneal amyloidosis with trichiasis.

Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms.

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.

Substance P-induced cadherin expression and its signal transduction in a cloned human corneal epithelial cell line.

Although the absence of Substance P (SP), a neurotransmitter in the trigeminal nerve, has been speculated as a cause for developing neurotrophic keratitis, its exact pathogenesis is still not clarified. In a previous report, we showed with electron microscopic examination that epithelial cell attachment was weakened in denervated corneas. In this study, SV40-transformed human corneal epithelial cells (HCE-Ts) were used to explore the molecular mechanisms responsible for mediating regulation of E-cadherin expression in response to Substance P receptor stimulation. Expression of the mRNAs for specific SP receptors, neurokinin (NK)-1R, NK-2R, and NK-3R, was demonstrated with RT-PCR. The cells were treated with various concentrations of SP in vitro, and the expression of an adhesion molecule E-cadherin was analyzed by immunofluorescence, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) using an anti-E-cadherin antibody. E-cadherin expression was increased by SP in a dose-dependent manner both in the cytosolic fraction and in the cell membrane fraction. This increase in E-cadherin expression was completely inhibited by Calphostin C (PKC inhibitor) and KN-62 (CaMK inhibitor), but not by H-89 (PKA inhibitor), indicating that SP-induced E-cadherin expression involves the activation of protein kinase C (PKC) and calmodulin kinase (CaMK). SP did not affect cell proliferation at all. All these findings indicate that SP induced E-cadherin expression through PKC and CaMK activation and suggest that a lack of SP may account in part for the pathogenesis of neurotrophic keratitis.

Effect of norepinephrine on proliferation, migration, and adhesion of SV-40 transformed human corneal epithelial cells.

PURPOSE: To determine the ability of norepinephrine to modulate proliferation, adhesion, and migration of SV-40 transformed human corneal epithelial cells. METHODS: Assays were performed using SV-40 transformed human corneal epithelial cells. For proliferation assays, cells were plated in 96-well plates coated with fibronectin and collagen (FNC). A dose-response curve was generated for norepinephrine in concentrations of 100 nM-100 microM. The cell number in each well was evaluated using the fluorochrome Calcein AM (an intracellular esterase cleavage substrate), and fluorescence was determined using an automated fluorescent plate reader. For cell adhesion, 25 x 10(-3) cells were plated onto FNC-coated 96-well plates, incubated in 10 nM-100 microM norepinephrine for 90 min, gently irrigated, and the remaining adherent cells quantitated. Cell migration was measured using blind-well migration chambers with a 10-microm pore size and FNC-coated filters. Cells (250 x 10(3)) were added to the upper chamber, incubated for 18 h in the presence of factors, after which time the cells that had migrated through the filter were quantitated. The toxicity of norepinephrine was evaluated using a standard Live/Dead assay employing the combined fluorochromes of ethidium homodimer (to indicate dead cells) and Calcein AM (to indicate viable cells). Varying concentrations of norepinephrine were added, and the cells incubated for 3 h and the fluorometric assay performed. RESULTS: Norepinephrine stimulated corneal epithelial cell proliferation and migration over a wide range of concentrations. It did not modulate cell adhesion and demonstrated cell toxicity only at the highest (supraphysiologic) concentration tested. CONCLUSIONS: Norepinephrine is normally found in the cornea and may be important in the maintenance of normal corneal homeostasis and in wound-healing processes.

Evaluation of cytotoxicity of various ophthalmic drugs, eye drop excipients and cyclodextrins in an immortalized human corneal epithelial cell line.

PURPOSE: An immortalized human corneal epithelial cell line (HCE) was tested as a screening tool for prediction of topical ocular irritation/toxicity by pharmaceuticals METHODS: Effects of various drugs, excipients and cyclodextrins (CDs) on viability of HCE cells were evaluated using two in vitro cytotoxicity tests, 3-(4,5-dimethlthiazol-2-yl)-205-diphenyl tetrazolium bromide (MTT) dye reduction assay and propidium iodide assay. RESULTS: Mitochondrion-based MTT test was a more sensitive indicator of cytotoxicity than the plasma membrane-based propidium iodide test. The tests revealed following cytotoxic rankings for ophthalmic drugs: dipivefrin > timolol > pilocarpine approximately equal to dexamethasone; for excipients: benzalkonium chloride (BAC) > sodium edetate (NA2 EDTA)>polyvinyl alcohol (PVA) > methylparaben; and for CDs :alpha- CD > dimethyl beta-cyclodextrin (DM-beta-CD) > sulfobutyl ether beta-cyclodextrin ((SBE)7m-beta-CD approximately equal to hydroxypropyl-beta-cyclodextrin (HP-beta-CD) > lambda CD. In consideration of the in vivo clinical situation, the short exposure time (5 min) is more relevant even though toxic effects of some test substances were seen only after longer exposure time (30 and 60 min). CONCLUSIONS: Immortalized HCE cells are a promising tool for rapid cytotoxicity assays of ocular medications. The cell line is potentially useful in predicting the in vivo coreal toxicity of ocularly applied compounds.

Neutrophil chemotaxis induced by corneal epithelial cells after herpes simplex virus type 1 infection.

PURPOSE: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.

Human corneal epithelial cells reorient and migrate cathodally in a small applied electric field.

PURPOSE: To test whether human corneal epithelial cells (HCECs) respond to small applied electric fields (EFs) in a similar manner to bovine corneal epithelial cells (BCECs), the orientation and directed migration in small EFs of both primary cultures and of a human corneal epithelial cell line were quantified. METHODS: Primary cultures of human corneal epithelial cells (PHCECs) and transformed human corneal epithelial cells (THCECs) were exposed to EFs (100 mV/mm-250 mV/mm) in different media. Cell migration was traced using an image analyser. RESULTS: PHCECs and THCECs reoriented and migrated towards the cathode (negative pole) when cultured in small direct current (dc) EFs. Both the reorientation and directional migration were voltage- and serum-dependent, as shown previously for bovine cells. PHCECs and THCECs showed significant perpendicular orientation in EFs at 150 mV/mm in medium with serum, while at the same voltage, no significant orientation was found in serum free medium. PHCECs started to show perpendicular reorientation around 30 min after onset of EF at 150 mV/mm. They showed significant directional migration at 150 mV/mm, with directedness of 0.35 +/- 0.07 and a migration rate of 9.1 +/- 0.7 microns/h (n = 90), both significantly higher than that of cells in serum free medium. Addition of EGF-induced significant reorientation and directional migration of THCECs at 100 mV/mm. Additionally, as for BCECs, which remained viable and responsive to electric fields for at least 75 h at 150 mV/mm, THCECs also remained viable and showed responsiveness during long periods of exposure to EFs (at least 20 h). CONCLUSIONS: Cultured human primary CECs and a human corneal epithelial cell line both responded to small EFs with perpendicular reorientation and cathodally-directed migration. Cell responses were qualitatively similar to those reported previously for bovine CECs. The endogenous EFs generated by wounded cornea may play an important role in promoting cell shape changes and directed migration of CECs during the healing process.

Human hepatocyte growth factor (HGF) in the aqueous humor.

Using an enzyme-linked immunosorbent assay, we measured the concentration of hepatocyte growth factor (HGF) in human aqueous humor and serum from 36 eyes of 33 patients and analyzed the relationship between HGF and several parameters of corneal endothelial cells. Aqueous HGF concentrations ranged from 0.020-0.194 ng/mL (average: 0.101 +/- 0.044 ng/mL) and was correlated positively with corneal endothelial cell density (r = 0.343, P = 0.04). The aqueous HGF level did not correlate with other corneal endothelial cell parameters or the serum HGF level. The HGF receptor, c-Met, was found in the corneal endothelial cell membranes. This study suggests that the aqueous humor HGF is a factor which maintains an integrity of corneal endothelial cells.

Effect of carbachol on phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis, and its modulation by isoproterenol in rabbit corneal epithelial cells.

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation by isoproterenol were investigated in SV40-adenovirus transformed rabbit corneal epithelial cells (RCEC). When examined under light microscope, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0.1 mM) for 30 min to RCEC, prelabeled with 32Pi, decreased the radioactivity in phosphatidylinositol 4-phosphate and PIP2 by 15 and 27%, respectively, and concomitantly increased the radioactivity in phosphatidylinositol and phosphatidic acid by 14 and 38%, respectively. When the concentration of CCh was increased to 1 mM, the changes in radioactivity were even more pronounced. Addition of CCh (0.1 mM) to the cells, prelabeled with myo[3H]inositol, increased the accumulation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3) by 115%, indicating stimulation of PLC-mediated PIP2 hydrolysis. Similar increases were also observed in [3H]InsP1 and [3H]InsP2. The effects of CCh on inositol phosphate accumulation were time- and dose-dependent, and were inhibited by atropine (10 microM), suggesting that the observed effects of CCh were mediated by activation of muscarinic cholinergic receptors. The effects of CCh were antagonized more potently by 4-diphenylacetoxy N-methyl-piperidine than by pirenzepine, indicating that the muscarinic receptors involved in PLC activation are probably of M3 type. By Western immunoblotting analysis with various anti-PLC antibodies, the RCEC were shown to contain PLC gamma 1 and PLC delta 1 in the soluble fraction and PLC beta 1 in the microsomal fraction. Addition of isoproterenol to RCEC, increased cAMP both in a time- and dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

An SV40-immortalized human corneal epithelial cell line and its characterization.

PURPOSE: The authors attempted to immortalize human corneal epithelial cells; it is difficult to propagate primary human corneal epithelial cells because of scarcity of available tissue. However, cell immortalization by virus is always accompanied by shedding of free virus. The current study was performed to establish a cell line that produces no free viral particle. METHODS: Primary cultured human corneal epithelial cells were infected with a recombinant sv40-adenovirus vector and were cloned three times to obtain a continuously growing cell line. Morphologic, cytologic, and biochemical characteristics of this cell line were analyzed. RESULTS: This cell line continued to grow for more than 400 generations, exhibiting a cobblestone-like appearance similar to normal corneal epithelial cells in culture. Transmission electron microscopy showed the evidence for the characteristic features of epithelial cells, including desmosome formation and development of microvilli. It expressed cornea-specific, 64-kD cytokeratin in addition to five major insoluble proteins. By enzymatic analysis using NADP as a coenzyme and a gas chromatograph mass spectrometer, this cell line was found to possess 8.71 IU/mg protein of aldehydedehydrogenase activity. When this cell line was grown at air-liquid interface on collagen type I gel, it differentiated in a multilayered fashion. CONCLUSIONS: The authors have established an SV40-immortalized human corneal epithelial cell line with properties similar to normal corneal epithelial cells.