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.

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

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.

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

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.

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.

Injection of chemoattractants into normal cornea: a model of inflammation after alkali injury.

PURPOSE: The objective of this study was to establish and characterize the invasion of polymorphonuclear leukocytes (PMNs) into a normal cornea after intrastromal injection of the tripeptide chemoattractants generated from alkali-degraded corneas. METHODS: The following samples were injected into the midstroma of normal rabbit corneas: ultrafiltered tripeptide chemoattractants (N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline) generated from alkali-degraded corneas, synthetic N-acetyl-PGP, positive control (leukotriene B4 [LTB4]), or negative control (Hanks' balanced salt solution [HBSS]). Timed responses of PMN infiltration were established for effective concentrations of LTB4 or the ultrafiltered chemoattractants. RESULTS: All intrastromal injections resulted in the immediate development of an edematous disc that was 10 mm in diameter. The lesion essentially had cleared in the HBSS-injected eyes by 8 hours, and histologic sections revealed minimal numbers of PMNs in the cornea or limbal tissue. The injection of LTB4 or the ultrafiltered tripeptide chemoattractants induced peak numbers of PMNs within the stroma at 8 hours, subsiding by 16 hours. Seventy units of ultrafiltered chemoattractants yielded a strong PMN response, similar to 1 X 10(-5) M LTB4. The highest concentration of ultrafiltered chemoattractants (350 U) produced a severe PMN response that was characterized by a solid sheet of neutrophils surrounding the injection site. The injection of synthetic N-acetyl-PGP (2 X 10(-4) M) produced a marked PMN response. CONCLUSIONS: PMN invasion of the normal cornea after the injection of the ultrafiltered tripeptide chemoattractants or the synthetic N-acetyl-PGP mimicked early PMN infiltration in the alkali-injured eye, confirming the importance of this chemoattractant as an inflammatory mediator.

A new classification system predicting keratomalacia after trauma in vitamin A deficiency: sodium citrate does not prevent disease progression.

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.

Effect of synthetic metalloproteinase inhibitor or citrate on neutrophil chemotaxis and the respiratory burst.

PURPOSE: A topical synthetic metalloproteinase inhibitor (SIMP) has been reported to reduce the incidence of corneal ulcerations in the alkali-injured eye by the putative mechanism of collagenase inhibition. The current study was performed to determine whether SIMP has a direct inhibitory effect on the activation of neutrophils and to compare any such effect with that of citrate, a known inhibitor of polymorphonuclear leukocyte (PMNL) activity. METHODS: The effect of SIMP or citrate was tested on chemotaxis of PMNL and the respiratory burst in vitro. RESULTS: Synthetic metalloproteinase inhibitor (1 mM) or citrate (12 mM) produced significant inhibition of chemotaxis of PMNL and the accompanying behavioral characteristics of motility. Synthetic metalloproteinase inhibitor, but not citrate, generated a dramatic respiratory burst when incubated with resting PMNL. Both SIMP and citrate inhibited the respiratory burst of PMNL in the presence of opsonized zymosan. For a brief, initial period each substance enhanced the respiratory burst generated by the metabolic stimulant from alkali-degraded corneas, followed by a rapid decline in activity that was associated with cell death. CONCLUSIONS: Although SIMP has been shown to exhibit powerful collagenase inhibition, its inhibitory effect on chemotaxis of PMNL might be the primary mechanism in reducing the incidence of ulceration in the alkali-injured cornea. Very significant reduction in the accumulation of PMNL in SIMP-treated corneas supports this conviction. Activation by SIMP of the respiratory burst in resting PMNL is of concern, but overall its beneficial effect is favorable, as demonstrated in prior alkali-injured animal models. The dual effect of inhibition of chemotaxis of PMNL and activity of collagenase makes SIMP a potential drug for combating ulceration in the alkali-injured cornea.

An excess of topical calcium and magnesium reverses the therapeutic effect of citrate on the development of corneal ulcers after alkali injury.

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.

A neutrophil chemoattractant is released from cellular and extracellular components of the alkali-degraded cornea and blood.

PURPOSE: A tripeptide chemoattractant(s) for neutrophils has been shown to release from alkali-degraded cornea. This study is designed to determine the source of the chemoattractant(s). METHODS: Whole corneas were degraded in 1 N NaOH for 10 minutes, 30 minutes, 1 hour, 4, 8, 24, and 48 hours to determine an optimal duration of alkali exposure for production of the chemoattractant. Whole cornea, cornea minus epithelium, cornea minus endothelium, and stroma alone were degraded in 1 N NaOH for 4 hours to determine the relative contribution of each corneal layer. In a separate experiment, epithelium alone, endothelium alone, cultured keratocytes alone, or bovine corneal collagen were treated separately in 1 N NaOH for 4 hours. Finally, human plasma, platelets, polymorphonuclear leukocytes (PMNL), and red blood cells were treated separately in 1 N NaOH for 4 hours to determine if a similar chemoattractant was released from alkali-treated noncorneal tissue. Neutralized suspensions of all samples were ultrafiltered and dialyzed. The chemotactic potential of each sample was determined in the polarization assay. RESULTS: Activation of the PMNL polarization response increased with the duration of exposure of corneal tissue to alkali, peaking at 6 hours. Release of the chemoattractant from alkali-degraded corneal tissue showed a significant decrease when the epithelium was removed from the stroma. All tissue layers showed a PMNL polarization response when treated with alkali. The response decreased from epithelium > endothelium > cultured keratocytes > collagen. Alkali degradation of human blood components, including plasma, showed significant polarization responses ranked in the following order: plasma > PMNL > tendon collagen > platelets = red blood cells. CONCLUSIONS: This study demonstrates that the tripeptide chemoattractant(s) is released from all layers of the cornea after alkali injury. The substance released from blood components is of similar size and biologic activity as that found in the cornea, but its exact molecular composition is yet to be determined. Timed response of alkali degradation determined the optimal conditions for generation of the chemoattractant(s) including clinically relevant time intervals.

Identification and synthesis of chemotactic tripeptides from alkali-degraded whole cornea. A study of N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline.

PURPOSE: To identify and synthesize the polymorphonuclear leukocyte chemoattractant(s) released from alkali-degraded corneas. METHODS: Corneas were degraded in 1.0 N NaOH, neutralized, ultrafiltered, and dialyzed. The final active ultrafiltrate was subjected to high-performance liquid chromatography on a Protein PAK I-60 column. The most active fractions were further separated on a mu-Bondapak-C18 and I-60 column in sequence. RESULTS: Fraction 38 from the final I-60 column associated with a 210-nm absorption peak and elicited a polarization and chemotactic response from polymorphonuclear leukocytes. The loss of polarization activity in fraction 38 after exposure to prolidase suggests that this peptide contains a Pro-X (X = amino acid) peptide bond. The amino acid composition of fraction 38 was 35% glycine and 53% proline. Peptide sequence analysis was unable to establish a primary sequence even though Picotag analysis showed the presence of large amounts of the two amino acids. Mass spectrometry revealed only two molecular species of 312 MWt and 284 MWt. Tripeptides were synthesized using all possible amino acid permutations of 2 Pro and 1 Gly and tested in the polarization and chemotactic assays. These techniques demonstrated that n-acetyl-Pro-Gly-Pro, and to a lesser degree n-methyl-Pro-Gly-Pro, were the only synthetic tripeptides with activity similar to the purified chemoattractant. CONCLUSIONS: The data show that the chemotactic peptides, purified from alkali-degraded whole cornea and confirmed with identical synthetic tripeptides, are N-acetyl-Pro-Gly-Pro and N-methyl-Pro-Gly-Pro. Although a number of proteins contain the Pro-Gly-Pro sequence, large amounts of collagen in the cornea suggest this as a major source. The small size and hydrophilic nature of these chemoattractants are predictive of a high degree of diffusibility. These chemoattractants are likely to play a major role in the early neutrophil response after an alkali injury.

Characterization of chemical gradients in the collagen gel-visual chemotactic assay.

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-degraded cornea generates a low molecular weight chemoattractant for polymorphonuclear leukocytes.

PURPOSE: The current study was designed to determine if a polymorphonuclear leukocyte (PMN) chemoattractant is derived from alkali-degraded whole cornea and to establish a range for its molecular weight. METHODS: We utilized a collagen gel-visual chemotactic assay to quantify the directional movement of PMN exposed to alkali-degraded corneas (30 min or 24 hr). In this experiment, the sample to be tested for chemotactic activity passed through a 14,000 molecular weight cutoff membrane into a collagen gel in which resting neutrophils were suspended in a random fashion. Cell movement was videotaped and subsequently tracked by digitizing the centroid of the cells at 30-sec intervals. Computer analysis of these tracks illustrated many behavioral characteristics, including directional movement. RESULTS: Alkali-degraded whole bovine corneas produced a chemotactic response in neutrophils within 5 min. Dilution of the 24-hr sample to 1:7 yielded a significant chemotactic response. The chemotactic response of both the 30-min and 24-hr samples followed a dose-response curve. CONCLUSIONS: This agent may be one of the inflammatory mediators that trigger the early neutrophil response after an alkali-injury to the eye.

Alkali-degraded cells generate a respiratory burst stimulant for neutrophils.

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.

Subconjunctival injection of purified blood mononuclear cells into alkali-injured rabbit eyes.

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.

A visual assay for quantitating neutrophil chemotaxis in a collagen gel matrix. A novel chemotactic chamber.

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.

Amino acid composition of a neutrophil respiratory burst stimulant. Evidence for a protein, noncollagenous source.

Activation of the neutrophil respiratory burst by the supernatant fraction from an alkali-treated collagen preparations (SAC) was enhanced by longer durations of exposure to alkali (1 N NaOH for 0.5-24 hr). The concentrate obtained from ultrafiltration (greater than 30,000 molecular weight) of SAC (1 N NaOH for 24 hr) retained the stimulatory factor. Fractionation of this ultraconcentrate by high-performance liquid chromatography showed that the stimulatory activity resided in the void volume (highest molecular weight). The amino acid composition of this active fraction revealed that this proteinaceous stimulant was not derived from the collagen molecule. Treatment of the SAC with ultrapure bacterial collagenase increased its stimulatory capacity, confirming its noncollagenous nature. Alkali treatment of whole cornea also released a similar large molecular weight, noncollagenous protein that stimulated the respiratory burst of polymorphonuclear leukocytes. Enhanced stimulation after prolonged NaOH treatment of the collagen preparation or collagenase treatment of SAC suggests that the stimulant might reside between collagen fibrils and then be released as the matrix is degraded.

The combined effect of citrate/ascorbate treatment in alkali-injured rabbit eyes.

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)

Topical citrate inhibits the adherence of neutrophils to postcapillary venules.

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.