Inhibitory effect of a complementary peptide on ulceration in the alkali-injured rabbit cornea.

PURPOSE: Two tripeptide chemoattractants, acetyl-proline-glycine-proline (Ac-PGP) and methyl-proline-glycine-proline (Me-PGP), are the primary triggers for early neutrophil invasion into the alkali-injured cornea. In the present study the effectiveness of a complementary peptide designed to inhibit the PGP chemoattractants (arginine-threonine-arginine [RTR] tetrameric peptide) and an apo A-1 mimicking peptide (5F) was investigated in the alkali-injured rabbit eye. METHODS: (L)-RTR tetramer, (D)-RTR tetramer, and 5F were tested in vitro for their effects on neutrophil polarization. Synthetic 5F was also tested in vitro for its effect on the neutrophil respiratory burst. In the alkali-injured rabbit eye model, the right corneas of 48 rabbits were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of three groups: phosphate-buffered saline (PBS) control; 800 microM RTR (dextrorotatory) tetramer in PBS alternating each hour with 1.5 mM RTR (levorotatory) tetramer in PBS; and 12 microM 5F in PBS. One topical drop of each substance was administered hourly (14 times per day) for 33 days. The experiment was continued until day 42 with no additional drops administered. RESULTS: (L)-RTR tetramer and (D)-RTR tetramer inhibited neutrophil polarization activated by the PGP chemoattractants in vitro. Synthetic 5F did not inhibit neutrophil polarization in the presence of Ac-PGP or the respiratory burst of neutrophils in the presence of a metabolic stimulant derived from alkali-degraded corneas. During the entire animal experiment, statistically fewer ulcers occurred in the RTR tetramer group than in the PBS control group (43.8% vs. 87.5%, P = 0.0046). The frequency of ulceration in the 5F group (68.8%) was not significantly different from the PBS control group. CONCLUSIONS: The reduction in the frequency of corneal ulceration by the RTR tetramer possibly resulted from its complementary binding to Ac-PGP and Me-PGP in the cornea shortly after alkali injury, leading to a reduction in the early and late infiltration of neutrophils. RTR tetramer appears to hold enough promise to warrant additional study as a therapeutic drug for the alkali-injured eye.     

L-arginine-threonine-arginine (RTR) tetramer peptide inhibits ulceration in the alkali-injured rabbit cornea

PURPOSE: Proline-glycine-proline (PGP) peptides have been identified as inflammatory mediators initiating neutrophil invasion into alkali-injured cornea. The complementary peptide, arginine-threonine-arginine (RTR), has been shown to bind to the PGP sequence and impede neutrophil infiltration. A prior study showed that L-RTR tetramer and D-RTR tetramer, used alternately (14 times a day), resulted in significantly reduced incidences of corneal ulceration and severity. The purpose of this experiment is to determine the effectiveness of both tetramers, used separately, compared with control. METHODS: Rabbit corneas were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of 3 groups: 1) phosphate-buffered saline (PBS), 2) 1.5 mM L-RTR, or 3) 800 microM D-RTR. One drop of each was administered hourly (14 times a day) for 36 days. Additional studies were done to assess neutrophil infiltration into corneas with and without RTR treatment. RESULTS: The severity of corneal ulceration in both RTR groups was statistically significantly different from the 21st day of the experiment to the end. As a result of ulcers healing in the L-RTR group, there was a statistically significant reduction in the number of ulcers beginning on day 22 versus control. Although there was healing in the D-RTR group, the incidence of ulcers was not significantly different from control or L-RTR. Morphometric analysis revealed decreased neutrophil (PMN) invasion with RTR treatment compared with PBS control. CONCLUSIONS: Binding of the PGP molecules by RTR tetramer seems to deprive the cornea of this neutrophilic chemotactic stimulus, leading to a reduction in the severity and incidence of corneal ulceration.     

Bioactivity of peptide analogs of the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

PURPOSE: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP. RESULTS: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition. CONCLUSIONS: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.     

High levels of apolipoproteins found in the soluble fraction of avian cornea

Water-soluble proteins in avian corneas were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Comparative protein profiling of avian and mammalian corneas revealed five major protein spots specifically detected in avian species. These proteins were identified as apolipoproteins A1 and D by tandem mass spectrometry sequencing. This is the first report of the presence of apolipoproteins in avian cornea. These results could provide insight into the role of lipid metabolism in the avian-specific function of cornea.     

Linkage of IL-6 with neutrophil chemoattractant expression in virus-induced ocular inflammation

PURPOSE: Herpes simplex virus (HSV)-1 infection of the murine cornea is known to stimulate a vigorous interleukin (IL)-6 response, but whether this pleiotropic cytokine is an essential participant in corneal inflammation is unclear. This study was designed to compare the early inflammatory response in IL-6 gene-deficient mice to that in wild-type hosts. METHODS: Gene knockout and wild-type mice (C57BL/6 background) were infected intracorneally with HSV-1 (strain RE) and observed through clinical examination and immunohistochemistry for the development of corneal opacity. Virus corneal titers were determined by standard plaque assay on Vero cells. Cytokine and chemokine levels in corneal lysates were measured with commercial ELISA kits. RESULTS: Corneal opacity in IL-6(-/-) mice was substantially diminished in comparison with IL-6(+/+) hosts 24 to 48 hours after intracorneal viral infection, and corneal levels of (MIP)-2 and MIP-1alpha were significantly reduced. Local administration of IL-6 at the time of infection restored corneal opacity and chemokine levels to that of wild-type hosts. Antibody neutralization of endogenous IL-6 in IL-6(+/+) animals reduced corneal opacity scores and MIP-2 levels to that of IL-6(-/-) mice. Ex vivo studies with excised corneal buttons revealed that uninfected IL-6(-/-) corneas injected with IL-6 produced MIP-2 and MIP-1alpha at levels comparable to that seen in IL-6(+/+) hosts. CONCLUSIONS: Collectively, these results suggest that IL-6 promotes corneal inflammation by acting in an autocrine-paracrine fashion to induce resident corneal cells to make MIP-2 and MIP-1alpha, which in turn recruit neutrophils to the virus infection site.     

The effects of hypertonic salicylate on neutrophil migration and vascularization in the rat cornea

Thermal cauterization of the center of the rat cornea results in emigration of neutrophils into the extravascular limbal tissue and blood vessel growth into the cornea. In this study, 1.0 M sodium salicylate, 1.0 M sodium chloride, and ointment vehicle were administered to normal and cauterized rat corneas for periods of 6, 48, and 144 h. When applied to the normal cornea, salicylate resulted in a marked increase in neutrophils in the limbal tissue at 6 h, but an inhibition at 48 h. Similarly, for the cauterized corneas, administration of salicylate increased the extent of neutrophil emigration at 6 h, but this effect was not sustained at 48 h. Neither vehicle nor sodium chloride had any effect on the extravascular neutrophil population. After 6 days, administration of the vehicle resulted in a slight increase in vascular growth into the cornea, whereas sodium salicylate caused a decrease. These findings indicate that hypertonic (1 M) sodium salicylate does not inhibit the emigration of neutrophils from limbal vessels of cauterized rat corneas, but does appear to have a cytotoxic effect on the tissues and on blood vessel endothelial cells.     

Dendritiform cells found in central cornea by in-vivo confocal microscopy in a patient with mixed bacterial keratitis

PURPOSE: To report the in-vivo confocal microscopic findings of dendritiform cells in the central corneal epithelial layer in a case of mixed bacterial keratitis associated with severe immunologic reaction. DESIGN: Observational case report. METHODS: A 25-year-old male suffered from contact lens-related mixed bacterial keratitis with a dense eccentric immune ring. In-vivo confocal microscopy was performed to study the different layers of the central and peripheral cornea in the lesion and the fellow eye. RESULTS: Several dendritiform cells were found in the basal and superficial epithelial layers of the central cornea in the lesion eye, which was also the area of the dense immune ring formation. No such cells could be identified in the fellow eye or the unaffected area of the lesion eye. CONCLUSION: Corneal dendritiform cells, possibly dendritic or Langerhans cells, can be identified in severe corneal immunologic conditions using in-vivo confocal microscopy. The role of these cells in ocular immunity is interesting and needs further clarification.     

Nanoparticles sustain expression of Flt intraceptors in the cornea and inhibit injury-induced corneal angiogenesis

PURPOSE: To determine whether long-term expression of intraceptors can be achieved using plasmid albumin nanoparticles and whether nanoparticles can inhibit and cause regression of murine corneal neovascularization induced by mechanical-chemical trauma. METHODS: Albumin nanoparticles encapsulating pCMV.Flt23K were developed as a lyophilized product that is easily redispersed in an aqueous medium. Nanoparticles were injected into the corneas of uninjured BALB/c mice and observed for toxicity for 3 weeks. Entry of nanoparticles into corneal cells was demonstrated through transmission electron microscopy and confocal imaging. Naked pCMV.Flt23K, nanoparticles encapsulating pCMV.Flt23K, or empty pCMV nanoparticles were injected into uninjured mouse corneas. These corneas were subjected to mechanical alkali trauma 3 weeks after injection. RESULTS: Nanoparticles were nontoxic to the cornea and entered into corneal keratocyte cytoplasm. They persisted for at least 4 weeks in the cornea, expressed effective intraceptor levels for at least 5 weeks, and reduced corneal neovascularization by approximately 40% (P = 0.035) at 5 weeks after administration. CONCLUSIONS: Albumin nanoparticles are not toxic to the cornea and can express intraceptors for extended periods that are effective in suppressing injury-induced corneal neovascularization.     

Electrolytes in the cornea: a therapeutic challenge

Background: Reported here are the results of electrolyte measurements in different layers of 70 apparently normal human corneas. Methods: Samples were examined by energy-dispersive X-ray analysis under calibrated conditions in a scanning electron microscope. The method allows the simultaneous quantitative analysis of, among others, sodium (Na), chloride (Cl), phosphorus (P) and potassium (K). The results are related to the dry weight of the analyzed samples. Four distinct layers, subepithelium, middle stroma, posterior stroma and Descemet's membrane, were analysed in each cornea. Results: In the middle stroma we found concentrations of: sodium 0.609 ± 0.13, chloride 0.557 ± 0.115, potassium 0.058 ± 0.02 and phosphorus 0.038 ± 0.01 (mmol/kg dry weight). Conclusion: The collation of normal electrolyte concentrations provides reference values for future studies on changes of the corneal electrolyte composition in diseased or injured eyes. The electrolyte composition of rinsing fluids or eye drops should be adjusted to that of the corneal stroma. Phosphate buffer, for example, is not a good vehicle for topical eye treatments and should be replaced by organic buffering systems.     

Demonstration of a protease inhibitor in the cornea.

Sequential extraction of bovine corneal homogenates with aqueous 0.154M NaCl, 0.5M NaCl and 3M guanidine HCl revealed the presence, in the two sodium chloride extracts, of trypsin inhibitory factors. Upon gel-filtration chromatography of the o.5M NaCl soluble corneal material on Sephadex G-75, two peaks with trypsin inhibitory activity were resolved. One peak was eluted in the void volume, whereas a second peak had mobility corresponding to a molecular weight fraction much lower than, and therefore distinct from, alpha 1-antitrypsin inhibitor. The possible implication of this inhibitory factor in the pathogenesis of corneal ulceration is briefly discussed.     

Oxygen tension distributions in the cornea: a re-examination

Previous calculations of the steady-state oxygen distribution in the in vivo cornea were made using assumed values for the needed parameters. Recent work has yielded experimental results on these quantities and we have used these to compute the oxygen tension distributions in the cornea for several conditions. The main result, which differs from the earlier calculations, is that there is an oxygen flux from the aqueous into the cornea in the open-eye steady-state condition. Oxygen tension profiles are also presented for the closed eye and for a corneal-contact lens system. Implications of the various profiles are discussed.     

Expression of the focal adhesion protein PINCH in normal and alkali-injured corneas and the role of PMNs

PURPOSE: To evaluate the role of particularly interesting new cysteine-histidine-rich protein (PINCH) in corneal wound healing and early neovascularization and to assess the influence of granulocytes. METHODS: A standardized corneal alkali wound was inflicted under general anaesthesia to the right eye of 14 New Zealand White rabbits. Seven of the rabbits received i.v. 5 mg/kg fucoidin every 2 hours to prevent granulocytes from entering the wound area. After 36 hours, the rabbits were killed, the corneas excised, fixed in 4% formaldehyde and embedded in paraffin. The sections were double-stained with antibodies against PINCH and with haematoxylin. RESULTS: In the normal cornea and limbus, PINCH was weakly expressed in the corneal epithelium and in a wedge of the conjunctival stroma. In the wounded corneas, PINCH expression was seen in the frontline of repopulating endothelial and epithelial cells, and in active keratocytes. The vascular endothelium and the granulocytes expressed PINCH, as did the conjunctival epithelium. In the fucoidin-treated rabbits, PINCH expression was markedly reduced. The vascular endothelial cells and the few granulocytes did not express PINCH in these rabbits. CONCLUSIONS: PINCH is only slightly expressed in the normal cornea. A corneal wound induces PINCH expression in the repopulating cells, in the vascular endothelial cells of the limbus, in the limbal epithelium and in the granulocytes. Exclusion of granulocytes reduces expression of PINCH and there is no expression at all in the vascular endothelium.     

Immunoprotein deposition in the cornea

A 63-year old woman had bilateral, multi-level corneal deposits distributed as fine, discrete crystals and in dense, deep geographic patches. She had a long history of sero-positive rheumatoid arthritis. Autopsy revealed an unsuspected lymphoproliferative disorder and immune-complex disease. Histologic examination of the eyes revealed eosinophilic, PAS-positive, noncollagenous deposits in the cornea at all levels and also in the ciliary processes, pars plana, and choroid. Stains for gold, amyloid, and acid mucopolysaccharides were negative. Immunoperoxidase stains were positive for IgG most strongly, and also for IgA, kappa and lambda light chains. Transmission electron microscopy showed needle-like electron-dense extracellular particles which we presume are immunoglobulins.