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Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

PURPOSE We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Our purpose was to determine whether chelation of Ca2+ and Mg2+ is the mechanism by which sodium citrate inhibits corneal ulceration in the alkali-injured rabbit eye. The right eyes of 60 albino rabbits (2-2.5 kg) were alkali-injured by filling a 12-mm-diameter plastic well placed on the corneal surface with 0.4 ml of 1 N NaOH. After 35 s the alkali was aspirated, and the well was rinsed with physiological saline. Animals were randomly distributed to three treatment groups of equal size. Two drops of the following topical medications were administered on the hour (14 times per day) for 35 days: physiological saline, 10% citrate in saline, and 346 mM Ca2+, 346 mM Mg2+, and 10% citrate in saline. During the experiment, significantly fewer ulcerations occurred in the citrate-treated eyes (five of 20, 25%) than in the saline-treated eyes (13 of 20, 65%) or in the calcium-magnesium-citrate-treated eyes (15 of 20, 75%). When ulcerations did develop in the citrate group, they occurred significantly later and were less severe than those in the saline and calcium-magnesium-citrate groups. There was a significant increase in the number of eyes with signs of band keratopathy and translucent areas in the calcium-magnesium-citrate group when compared with the other two groups. As in previous studies, sodium citrate significantly inhibited the development of corneal ulcers after alkali injury. The annullment of the favorable effect of citrate on ulceration in the alkali-injured eye by the addition of calcium and magnesium shows that the mechanism of action of citrate is the chelation of these divalent cations.

Chemical gradients developing in a collagen gel-visual chemotactic assay (CG-VCA) for PMNs were evaluated by theoretical and experimental methods. First, a video image analysis system was used to establish the diffusion coefficients of bromophenol blue (BPB) through the membrane (D1) and across the collagen gel (D2) in a capillary tube apparatus used only for this purpose. The diffusion coefficients of BPB and the geometry of the CG-VCA system were then used to develop a mathematical model, estimating theoretical gradients in the CG-VCA system. In addition, gradients in the CG-VCA system were characterized experimentally by employing BPB as the source and determining the concentration profiles of BPB in the chemotactic compartment by video image analysis. A relative error of approximately 21% exists between the theoretical and experimental gradients for both 0.5 and 1.0 mM source concentrations of BPB. This favorable comparison demonstrates reliability in predicting chemical gradients for the CG-VCA system. The mathematical model was then used to predict gradients using nanomolar and micromolar concentrations of low molecular weight chemoattractants. Analysis of these specific gradients showed that gradients were steep enough to be detected by PMNs in the collagen gel during the observation period used in previous experiments. The determination of BPB gradients in the CG-VCA system illustrates the utility of this system.

Alkali treatment of whole cornea produces a high molecular weight polypeptide causing a respiratory burst in neutrophils. The current study was conducted to determine if this stimulant could be elicited from cultured or scraped corneal cells (epithelium, keratocytes, or endothelium). Fresh or previously frozen epithelium, endothelium, and stroma were isolated from bovine eyes, alkali treated, and tested for the presence of the respiratory burst stimulant. The same procedure was performed on cultured bovine epithelium, keratocytes, and endothelium. To determine if the stimulant is specific to corneal cells, the component parts of human blood (cellular and extracellular) were separated, treated with alkali, and tested individually. Activation of the respiratory burst of polymorphonuclear leukocytes (PMNs) was used as a marker for the presence of the stimulant. All alkali-degraded cell samples, except red blood cells, produced a respiratory burst when added to viable PMNs. Alkali-treated plasma induced weak stimulation. Bovine corneal epithelium was alkali-treated and separated by high-performance liquid chromatography (HPLC) according to molecular size. The stimulant was shown to have a high molecular weight by its recovery in HPLC fraction number 7. These results demonstrate that the PMN stimulant originates from cells. This PMN stimulant is proposed as an inflammatory mediator in the alkali-injured cornea.

The chemotactic behavior of polymorphonuclear leukocytes (PMNs) suspended in a three-dimensional gel of native collagen fibers was analyzed using a new visual assay aided by computer assisted tracking. Cell behavior was observed in a 7 microliters chamber closed at either end with capillary tubes tipped with dialysis membrane. The chemoattractant, LTB4, was placed in one capillary tube and the control substance in the opposite tube. Under microscopic observation neutrophils were videotaped, their images digitized, and the x and y coordinates of the cell centroids captured at 30 s intervals for 15 min and subsequently analyzed. The data generate a global perspective of neutrophil behavior in a medium simulating a collagenous tissue. The results show that when leukotriene B4 was substituted for HBSS the PMN population underwent chemotactic displacement. PMN chemotaxis was increased greatly when the concentration of LTB4 was increased from 10 nM to 1 microM in separate experiments. This result was partly achieved by movement of an increasing percentage of the PMN population, less frequent stops, and longer durations of motility for individual cells. The most dramatic effect of LTB4 on neutrophil behavior was a large increase in directional movement toward the chemotactic source. The effects of LTB4 fell dramatically when the gradient source concentration was increased to 10 microM. The visual assay described here provides clear evidence that LTB4 induces true neutrophil chemotaxis in a collagenous matrix.

Monocytes transform into tissue macrophages that scavenge necrotic debris from wounds but also release substances stimulatory to fibroblasts. In an effort to encourage the healing process, monocytes, purified from rabbit blood, were injected subconjunctivally 1 or 2 days and 6 or 7 days after alkali injury to the rabbit eye. The injected monocytes failed to alter the course of the alkali-injured eyes. Specifically, the incidence of corneal ulceration in the experimental group was not statistically different from that of the control group. A byproduct of this study was a presentation of the technique to purify platelet-free mononuclear leukocytes from rabbit peripheral blood.

The incidence of ulceration and perforation in the cornea of alkali-injured eyes is significantly reduced by treatment with trisodium citrate or sodium ascorbate. Topical citrate reduces the inflammatory response in the cornea by inhibiting polymorphonuclear leukocytes. Topical ascorbate elevates the depressed level of this vitamin in the alkali-injured cornea, reversing a scorbutic condition. The purpose of the current study was to determine whether combined treatment with topical citrate and ascorbate has an additional therapeutic value when compared with citrate alone. Adsorbotear without EDTA was used as the vehicle for both medications. Rabbit eyes were injured with 1N NaOH for 35 s using a 12-mm well and were rinsed. Group I (47 eyes) received two drops of 10% citrate every hour on the hour and Adsorbotear on the half-hour for 14 h/day. Group II (48 eyes) received two drops of 10% citrate every hour on the hour and 10% ascorbate every hour on the half-hour for 14 h/day. The citrate/ascorbate group had significantly fewer ulcerations during the experiment than did the group receiving citrate alone (2 of 48 versus 10 of 47, 0.01 less than p less than 0.02). Both anterior ulcers in the citrate/ascorbate group and five ulcers in the citrate group healed to no ulcer by the end of the experiment (0 of 48 versus 5 of 47, 0.02 less than p less than 0.05). The average depth of ulceration was significantly less for the citrate/ascorbate group (p less than 0.001). No descemetoceles or perforations were observed in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

PURPOSE The purpose of this study was to develop a classification system to predict keratomalacia after trauma in vitamin A-deficient eyes and to determine whether citrate impedes polymorphonuclear leukocyte infiltration into the cornea, thus preventing keratomalacia. METHODS Preliminary classification studies showed that a 7.0-mm corneal epithelial scrape, before clinical findings of corneal xerosis, did not induce keratomalacia. Primary studies were conducted concurrently on the same animals to develop the classification system and test the effect of citrate in vitamin A deficiency. A 7.0-mm corneal epithelial scrape was performed on vitamin A-deficient eyes in various stages of corneal xerosis and treated as follows. Experiment 1: group 1, 10% citrate drops; group 2, phosphate buffer solution (PBS) drops; experiment II: group 3, drops and subconjunctival injection of 10% citrate; group 4, drops and subconjunctival injection of PBS. RESULTS Corneal abrasion in eyes with 2+ corneal xerosis yielded keratomalacia in 50% of cases; the remainder healed with xerotic epithelium. Eighty-three percent of eyes with > 2+ xerosis developed keratomalacia after corneal abrasion, whereas only 7.1% of eyes with < 2+ xerosis advanced to this stage. In experiment I, 27% of citrate-treated eyes and 38% of PBS-treated eyes developed keratomalacia (not significant). In experiment II, two of six citrate-treated eyes perforated and one eye developed keratomalacia. One of six control PBS eyes perforated and four developed keratomalacia. CONCLUSION We correlated the degree of corneal xerosis with the occurrence of keratomalacia after corneal trauma. This led to the development of a classification scale that is of research and clinical significance. Additionally, citrate did not significantly reduce keratomalacia or perforation in the vitamin. A-deficient eye.

The high incidence of corneal ulceration in the alkali-injured rabbit eye can be reduced by topical citrate drops. Citrate interferes with polymorphonuclear leukocyte (PMN) accumulation in the corneal stroma after alkali injury. It inhibits the adherence, locomotion, respiratory burst, phagocytosis, and degranulation of human PMNs in vitro. The objective of this research was to determine if PMN adherence to the vascular endothelium could be inhibited by superfused citrate in vivo. PMN adherence to the endothelium of postcapillary venules in the hamster cheek pouch was greatly enhanced and maintained by addition of leukotriene B4 (LTB4) to the superfusate. Superfusion of the pouch with 24 mM citrate for 5 min, before addition of LTB4, inhibited PMN adherence to the endothelium. The free calcium level in the superfusate was reduced from approximately 650 to 33 microM in the presence of 24 mM citrate. When LTB4-augmented PMN adherence was allowed to occur, followed by citrate treatment (24 mM), it was reversed. Simultaneous addition of CaCl2 and MgCl2 to the superfusate raised the free Ca2+ level to 786 microM and reversed the citrate-induced inhibition of LTB4-augmented adherence. From this and other research, we conclude that citrate probably inhibits adherence of PMNs to pericorneal and conjunctival vessels in the eye, preventing their accumulation in the corneal stroma.

Topical citrate has been shown to significantly reduce the severity and incidence of corneal ulceration in alkali-burned rabbit eyes. Subcutaneous injection of citrate significantly reduced the severity of ulceration. This study was performed to determine the plasma citrate and ionized calcium levels after systemic administration of citrate. Repeated ingestion of citric acid capsules showed a small increase in the plasma citrate level, but no significant change in the level of ionized serum calcium 30 min after the last ingestion. A single dose of Polycitra-LC (6.672 g of citrate) more than doubled the baseline citrate concentration but only induced a 5% decrease in the level of ionized serum calcium 30 min after ingestion. Neither regimen of orally administered citrate produced any significant effect on the respiratory burst or adherence of in vitro polymorphonuclear leukocytes (PMNs) resuspended in their respective plasma. In comparison studies, higher levels of plasma citrate and lower levels of ionized serum calcium were achieved through plateletpheresis. The respiratory burst and adherence of PMNs were not statistically different from baseline PMNs. However, there was an inverse correlation between the micromolar increase in citrate or the micromolar decrease in calcium and the respiratory burst. These results suggest that the use of oral citrate alone may not be practical, but does not preclude the use of oral citrate as a supplement to topical citrate.

This study was undertaken to demonstrate the effect of alkali on the molecular size of collagen. Type I and V collagen from bovine cornea were incubated with different concentrations of NaOH at room temperature and 37 degrees C for various times. The samples were then neutralized and analyzed by high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degradation of collagen molecules to a heterogeneous mixture of peptides with molecular weights less than 20,000 daltons occurred at concentrations greater than 0.25N, and the effect of alkali was much faster at 37 degrees C than at room temperature. In contrast, the molecular size of collagen was not affected by similar concentrations of HCl under similar conditions of time and temperature.