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Originally published In Press as doi:10.1074/jbc.M410638200 on February 11, 2005

J. Biol. Chem., Vol. 280, Issue 15, 15267-15278, April 15, 2005
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A Role for the Mouse 12/15-Lipoxygenase Pathway in Promoting Epithelial Wound Healing and Host Defense*{boxs}

Karsten Gronert{ddagger}, Neha Maheshwari, Nabeela Khan, Iram R. Hassan, Michael Dunn, and Michal Laniado Schwartzman

From the New York Medical College, Department of Pharmacology, Valhalla, New York 10595

Received for publication, September 15, 2004 , and in revised form, February 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The surface of the eye actively suppresses inflammation while maintaining a remarkable capacity for epithelial wound repair. Our understanding of mechanisms that balance inflammatory/reparative responses to provide effective host defense while preserving tissue function is limited, in particular, in the cornea. Lipoxin A4 (LXA4) and docosahexaenoic acid-derived neuroprotectin D1 (NPD1) are lipid autacoids formed by 12/15-lipoxygenase (LOX) pathways that exhibit anti-inflammatory and neuroprotective properties. Here, we demonstrate that mouse corneas generate endogenous LXA4 and NPD1. 12/15-LOX (Alox15) and LXA4 receptor mRNA expression as well as LXA4 formation were abrogated by epithelial removal and restored during wound healing. Amplification of these pathways by topical treatment with LXA4 or NPD1 (1 µg) increased the rate of re-epithelialization (65–90%, n = 6–10, p < 0.03) and attenuated the sequelae of thermal injury. In contrast, the proinflammatory eicosanoids, LTB4 and 12R-hydroxyeicosatrienoic acid, had no impact on corneal re-epithelialization. Epithelial removal induced a temporally defined influx of neutrophils into the stroma as well as formation of the proinflammatory chemokine KC. Topical treatment with LXA4 and NPD1 significantly increased PMNs in the cornea while abrogating KC formation by 60%. More importantly, Alox15-deficient mice exhibited a defect in both corneal re-epithelialization and neutrophil recruitment that correlated with a 43% reduction in endogenous LXA4 formation. Collectively, these results identify a novel action for the mouse 12/15-LOX (Alox15) and its products, LXA4 and NPD1, in wound healing that is distinct from their well established anti-inflammatory properties.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of an inflammatory/immune response is paramount for mucosal and dermal tissues, since they form a critical barrier to pathogens (1, 2). However, execution of a self-resolving inflammatory-reparative response is essential to maintain tissue function. This is especially relevant in the cornea, since it is a frequent site of infection, injury, and surgery; restoring its function is critical in order to preserve vision (3–10). The cornea shows a remarkable and highly regulated capacity for epithelial regeneration, unlike other tissues (11); it appears to be dependent on infiltration of polymorphonuclear leukocytes (PMN)1 into the wounded tissue (12). A cardinal sign of corneal injury or infection is the recruitment of leukocytes and formation of lipid and protein mediators that initiate and/or amplify inflammation and neovascularization (4, 5, 13–15). However, aberrant activation of these circuits can lead to sight-destroying inflammation and, thus, has been a main focus of research efforts. A striking feature of the avascular cornea is that it is largely devoid of immunogenic-inflammatory responses. Moreover, soluble factors from the aqueous humor appear to actively suppress leukocyte activation (7–10). However, endogenous mechanisms that mediate this atypical inflammatory response remain to be clearly delineated. No clinical therapies are currently available that selectively target inflammation or promote wound healing in the cornea. More importantly, use of anti-inflammatory glucocorticoids for corneal inflammation is restricted by serious side effects, including cataract formation, glaucoma, activation of latent herpes simplex, and delayed wound healing (16).

Lipoxygenase (LOX)-initiated lipid mediator pathways are important regulators of innate immunity and inflammation, since they can activate both pro- and anti-inflammatory signals (3, 17–20). Recent studies have demonstrated that human pathogens such as Pseudomonas aeruginosa and Toxoplasma gondii can exploit these pathways to suppress host immune responses by expressing a functional 15-LOX and inducing LXA4 formation (21–23). The human and murine 12/15-LOX pathways are of particular interest, since they are key enzymes in the formation of a distinct class of eicosanoids, the lipoxins, which are autacoids generated during inflammation and heterotypic cell-cell interactions. Lipoxin A4 exhibits potent anti-inflammatory properties in vitro and, in animal models of acute or aberrant inflammation, by inhibiting PMN (18) and lymphocyte activation (24) as well as suppressing dendritic cell function (21, 22). In this regard, LXA4 has been implicated as an important component of the endogenous counterregulatory signals that promote resolution of inflammation and limit PMN-mediated tissue injury (3, 18, 25). This conclusion has been further substantiated by recent studies with LXA4 receptor (ALX) transgenic mice, which demonstrate inhibition and accelerated resolution of inflammatory responses (26, 27).

The importance of mediators derived from essential polyun-saturated fatty acid is underscored by the well established role of {omega}-3 fatty acids in maintaining human health (28–30). Epidemiological studies have demonstrated that consumption of fish oil-enriched diets containing eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) lowers the incidence of inflammatory and autoimmune diseases (31). Docosahexaenoic acid (DHA, 22:6), in particular, is highly concentrated in photoreceptors, brain, and retinal synapses, and evidence suggests that it exerts anti-inflammatory and immunosuppressive effects (28, 32–34). However, until recently, no clear mechanism could account for these beneficial properties of DHA. It is of interest that several reports provide evidence for a 15-LOX-like initiated pathway that gives rise to a DHA-derived mediator (35–37), neuroprotectin D1 (NPD1; 10,17S-docosatriene), which mimics many of the anti-inflammatory actions of LXA4. Specifically, NPD-1 exhibits neuroprotective properties in experimental ischemic stroke and protects human retinal pigment epithelial cells from oxidative stress-induced apoptosis (35, 36). Although endogenous NPD-1 formation has been demonstrated in mouse brains, it remains to be determined whether this protective mediator can be generated in other tissues or organs.

It is notable that corneal epithelia of several species express prominent 12/15-LOX activity (38, 39). Furthermore, RNA expression of 15-LOX type 1 (ALOX15) and type 2 (ALOX15B) has been demonstrated in human corneal epithelial cells (40, 41), and analysis by immunohistochemistry suggests 12/15-LOX (Alox15) expression in mouse lens epithelial cells (42). However, no studies have identified a role for these enzymes in the cornea or investigated whether these pathways give rise to lipid autacoids that may regulate inflammatory or immunogenic responses in the cornea. To this end, we set out to determine whether anti-inflammatory lipid autacoids are formed in the cornea as part of an endogenous program to limit the sequelae of epithelial injury. Here, we report the endogenous formation of LXA4 and NPD1 in healthy and injured corneas and mRNA expression of a LXA4 pathway that correlates with epithelial wound healing. In addition, we provide evidence for a novel role of the mouse 12/15-LOX pathway in wound healing and demonstrate that treatment with LXA4 and NPD1 accelerates re-epithelialization without impairing PMN recruitment, a bioaction distinct from their documented leukocyte-selective properties.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Experiments
All animal studies were approved by the New York Medical College Vertebrate Animal Committee and were performed according to the guidelines of the Association for Research in Vision and Ophthalmology. Male BALB/c mice (6–8 weeks) were placed either on a standard rodent diet or on a custom diet (Research Diets, Inc.) that was based on either corn oil or fish oil (menhaden oil, {omega}-3) as a fat source. The AIN-76A diet is a standard low fat rodent diet that contains 20.8 kcal % protein, 67.7 kcal % carbohydrates, and 11.5 kcal % fat. The menhaden oil contains 2% arachidonic acid, 14% eicosapentaenoic acid, and 11% DHA and represents 90% of the fat in the {omega}-3 diet. The fish oil diet was kept at 4 °C in the dark and was replaced every 2 days, and animals were placed on the diet for 3–4 weeks prior to being subjected to corneal injury.

Mice were anesthetized with ketamine (50 mg/kg) and xylazine (20 mg/kg) intramuscularly, and a drop of tetracaine-HCl 0.5% was applied to the eye to deliver local corneal anesthesia prior to subjecting animals to two separate models of corneal injuries.

Thermal Injury—Corneas were cauterized by drawing a vertical line (~1–2 mm) across the cornea with a heated microprobe under a slit lamp biomicroscope. This method results in a significant and consistent injury, namely a 1–2-mm burn that was restricted to the anterior corneal surface. Wound size and degree of injury were determined by slit lamp biomicroscopy and documented for quantitation and analysis by a CCD camera (MicroPublisher, QImaging).

Epithelial Removal—The corneal epithelium up to the corneal/limbal border was removed using an Algerbrush II with a 0.5-mm corneal rust ring remover under the slit lamp biomicroscope. Corneas were stained with fluorescein as a direct marker of the epithelial defect, and the total area of the denuded cornea was quantitated by image software analyses.

Mice were treated topically (1 eye drop; 10 µl) immediately after thermal injury or de-epithelialization with 0.01–1 µg of the indicated lipid autacoids in 100% sterile HBSS (pH 7.4); solutions for every treatment were prepared immediately prior to the application. Topical treatments were repeated three times daily for 24–96 h. LXA4 was purchased from Calbiochem, and 17S-HDHA and NPD1 were prepared by biogenic synthesis. LXA4 stable analog (ATLa, 15-epi-16-(para-fluorophenoxylipoxin A4-methyl ester) was kindly provided by Drs. John F. Parkinson and William J. Guilford at Berlex Bioscience.

12/15-LOX (Alox15)-deficient mice (6 weeks, female) were purchased from Jackson Laboratory (Bar Harbor, ME). These mice are a Jax®Gemm® Strain with a targeted mutation in the 15-LOX (Alox15tm1Fun) that is in a background C57BL/6J inbreed strain. They are well characterized (43–45) and do not express the "leukocyte-type" 12/15-LOX (Alox15). Age- and gender-matched congenic C57BL/6J stock 000664 mice (6 weeks, female) were used as controls as suggested by Jackson Laboratory.

Assessment of Inflammation and Wound Healing
Mice were anesthetized, and corneas were subjectively assessed 24, 48, 72, and/or 96 h after injury for cloudiness, opacity, and neovascularization as markers of inflammation by slit lamp biomicroscopy, as previously cited (6, 14). Eyes of anesthetized mice were stained with fluorescein, and images of the anterior surface were taken with a CCD camera attached to the slit lamp biomicroscope to quantitate wound closure and re-epithelialization. Digital images were analyzed, and the fluorescein-stained wound area was quantitated using image software analyses (Adobe Photoshop, San Jose, CA).

PMN were quantitated in dissected and cleaned corneas at the indicated time points by measuring myeloperoxidase activity (MPO) as a specific PMN marker (25, 27, 37, 46). In brief, tissues were homogenized in potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide, followed by three cycles of sonication and freeze-thaw. The particulate matter was removed by centrifugation, and MPO activity in the supernatant was measured by spectrophotometry using o-dianisidine dihydrochloride reduction as a colorimetric indicator. Calibration curves for conversion of MPO activities to PMN number were established with PMN that were collected from zymosan A-induced peritonitis in BALB/c mice.

RT-PCR
Corneas were dissected from eyes using sterile instruments and were cleaned in sterile phosphate-buffered saline (4 °C) under a dissecting microscope to remove all noncorneal tissue. RNA was isolated using TRIzol reagent (Invitrogen), and RNA integrity was verified by agarose gel electrophoresis and quantitated by spectrophotometry. Specific primers were designed based on published mRNA sequences (PubMed, GenBankTM) to amplify the mouse lipoxin receptor ALX as previously described (47). The following established primer pairs were used to amplify mouse lipoxygenases: 1) leukocyte-type 12/15-LOX (Alox15) (48, 49) (sense, 5'-GCGACGCTGCCCAATCCTAATC-3'; antisense, 5'-CATATGGCCACGCTGTTTTCTACC-3'); 5-LOX (Alox5) (50) (sense, 5'-ATGCCCTCCTACACTGTCAC-3'; antisense, 5'-CCACTCCATCCATCTATACT-3'); epidermal-type 12-LOX (Aloxe) (51) (sense, 5'-AGTGACACCGATGTGAAGGAG-3'; antisense, 5'-CTCTCAGATGGTCACACTG-3'); platelet-type 12-LOX (Alox12) (sense, 5'-CGCGGGGCAAGGAGGAGGAGT-3'; antisense, 5'-GGGGTTGGCGCCATTGAGGA-3'). {beta}-Actin was used as a reference gene and amplified using the sense primer 5'-ACGGCCAGGTCATCACTATTG-3' and antisense primer 5'-AGGGGCCGGACTCATCGTA-3'. Two PCR amplifications were carried as controls for each primer pair and corneal RNA sample to confirm specific amplification: 1) without the RNA template and 2) without reverse transcription. RT-PCR analysis was performed with the OneStep RT-PCR kit (Qiagen, Valencia, CA) and a Gradient thermal cycler (PTC-200; MJ Research).

Lipid Autacoid Analysis
Eicosanoids and DHA products were analyzed employing reverse phase high performance liquid chromatography (RP-HPLC) and capillary gas chromatography/mass spectrometry (GC/MS) analysis as previously described (25, 27, 37, 46, 52). In brief, analysis was performed with an Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA) equipped with a diode array detector, a binary pump, a membrane degasser, a heated column compartment, and a microflow cell. Samples were injected in mobile phase and eluted on a Luna C18–2 microbore column (1 x 100 mm, 5 µm; Phenomenex, Torrance, CA) or a Beckman Coulter Ultrasphere column (4.6 x 250 mm, 5 µm; Phenomenex) using two separate gradient mobile phase systems that consisted of methanol/acetonitrile/water/acetate (24:27:49:1, v/v/v/v, pH 5.6) or methanol/water/acetate (65:35:0.01, v/v/v) run isocratically for 25 min and followed by a 25-min linear gradient to 99:1 or 99.99:0.01 (v/v, methanol/acetate), respectively. Collected UV data were recalled at 205, 236, 270, 245, 280, and 301 nm and analyzed by LC 3D ChemStation software (Agilent Technologies). Calibration curves were established for each compound, and peak areas were integrated for quantitation.

GC/MS analysis was performed as in Ref. 46 with a 6890N GC system with a HP5MS cross-linked ME siloxane column (30 m x 0.25 mm x 0.25 µm), an autosampler, and a 5973N mass-selective detector (Agilent Technologies). The helium flow rate was 1.5 ml/min, and the initial temperature was 150 °C, followed by 230 °C (2 min) and 280 °C (10 min). For selected samples, HPLC fractions that correspond to established retention times of authentic standards were collected and derivatized to generate pentafluorobenzyl esters and trimethylsilyl ether derivatives, and 10–1000 pg were injected in 2 µl of iso-octane for GC/MS analysis.

For endogenous product analysis, corneas from healthy or injured eyes were dissected in cold HBSS buffer without calcium or magnesium, cleaned under a dissecting microscope to remove all noncorneal tissue, and either immediately placed in 100% MeOH (injured corneas) or in HBSS buffer (pH 7.4, 37 °C) containing 1.2 mM Ca2+. Uninjured corneas in buffer were incubated in the dark (37 °C, 30 min) in the presence of a calcium ionophore (A23187 [GenBank] ; 5 µM). Prostaglandin B2 (50 ng) was added to samples as an internal standard for recovery, and corneas were gently homogenized at 4 °C and extracted as previously detailed (53). In brief, homogenized corneal suspensions were placed at -20 °C for at least 30 min and centrifuged, and supernatants were collected. Supernatants were diluted with 10 volumes of HPLC grade water and acidified to pH 4.0 with HCl (1 N). Acidified samples were immediately loaded onto primed C18-ODS cartridges (AccuBond II; 500 mg; Agilent Technologies). Cartridges were washed with 10 ml of HPLC grade water followed by hexane, and compounds were eluted in methyl formate followed by a final elution in methanol. Methyl formate fractions were taken to dryness under a gentle stream of nitrogen, resuspended in MeOH (100 µl), and stored at -80 °C.

To quantitate formation of lipid autacoids, extracted samples in methanol (methyl formate fractions) were taken to dryness under a gentle stream of nitrogen, resuspended in ELISA buffer, and immediately analyzed by specific ELISAs for LXA4 (54) (Neogen, Lexington, KY), PGE2, and LTB4 (Cayman Chemical, Ann Arbor, MI) according to the manufacturers' instructions.

To quantitate chemokine formation, corneas (2 corneas per data point) were isolated and homogenized on ice in HBSS buffer containing protease inhibitor mixture (Complete Mini; Roche Applied Science) without calcium or magnesium. Cell debris was removed by centrifugation, and supernatants were analyzed for mouse KC formation by Pierce, using a custom SearchLight quantitative multiplexed sandwich ELISA Proteome Array.

Biogenic Synthesis of NPD1
Reference material (17S-resolvins) and compounds for biological assays (17S-HDHA, 10,17S-diHDHA) were prepared by incubation of DHA (Cayman Chemical) and soybean LOX (Type IV) and/or 5-LOX from potato using different ratios of substrate to enzyme to give specific product profiles. Each reaction condition used was optimized as previously described (37, 46). Reactions were terminated by acidification and extracted, and products were isolated by RP-HPLC. Physical criteria for identification such as HPLC retention times, specific UV chromophores, and GC/MS and LC/MS major and signature ions have been established for 17S-resolvins (35, 37, 46). Endogenous arachidonic acid LOX products, such as LTB4, 5,12-diHETE isomers, 12S-HETE, 15S-HETE, and 5S-HETE, as well as standards for the docosanoids 7S,17S-diHDHA, 14S-HDHA, 7S-HDHA, and 4S-HDHA, which exhibit similar UV characteristics, had distinct retention times in our HPLC system and did not coelute with 17S-HDHA or NPD1.

Statistics
All data are expressed as means ± S.E. unless otherwise indicated. Significance of differences was determined by regression analysis and one-way ANOVA with STATISTICA© (version 6.1; StatSoft, Inc.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
An Anti-inflammatory Lipid Mediator Pathway Is Expressed in the Corneal Epithelium—We employed a semiqualitative RT-PCR analysis to examine the expression of leukocyte-type 12/15-LOX (Alox15) and LXA4 receptor (ALX) before and during injury using a model of epithelial injury that demonstrates defined wound healing (Fig. 1A) and PMN infiltration (Fig. 1B). Epithelial removal induced a consistent wound (6.81 ± 0.03 mm2, n = 46) that exhibited a linear rate of re-epithelialization (Fig. 1A) with 40 ± 3 and 88 ± 7% wound closure by 48 and 96 h (n = 4–12), respectively. This injury was associated with significant and transient PMN infiltration, measured by myeloperoxidase activity, into the cornea (Fig. 1B), which peaked at 48 h (33,097 ± 3948 PMN/cornea, n = 8) and decreased by 96 h (17,541 ± 8166 PMN/cornea, n = 8). Uninjured corneas had no detectable myeloperoxidase activity (Fig. 1B, n > 20). Hematoxylin/eosin staining of frozen 10-µm corneal sections confirmed both PMN infiltration into the stroma and re-epithelialization of the denuded cornea as well as the absence of PMN in uninjured corneas (data not shown). RT-PCR analysis demonstrated that normal cornea expressed both Alox15 and ALX receptor (Fig. 1, C and D) genes and that epithelial removal induced a marked decrease of ALX and Alox15 mRNA expression at 24 h (n = 4) after injury. mRNA expression of both genes returned to basal levels by 48–96 h (n = 4).



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  		FIG. 1.
An anti-inflammatory lipid mediator pathway is expressed in the corneal epithelium. Mouse corneas were collected at indicated times from eyes after epithelial removal (see "Experimental Procedures") or from healthy (uninjured) eyes (CT). A, temporal profile of re-epithelialization of mouse corneas after injury. The area of the denuded cornea was quantitated by fluorescein staining and digital image analysis (n = 4–12; symbols indicate differences at all data points, p < 0.017). B, epithelial removal induces temporally defined PMN infiltration. Corneas were collected, and PMN content was determined by measuring myeloperoxidase activity as a quantitative PMN marker (n = 8–10; *, different from uninjured, p < 0.04). C and D, RNA expression of LXA4 receptor and 15-LOX in healthy and injured corneas was analyzed by semiquantitative RT-PCR. The epithelium of the right cornea was removed, and the left cornea served as the uninjured or epithelialized control. Corneas were collected at the indicated time points, and isolated RNA was analyzed by specific primers for mouse L-12/15-LOX (C) or ALX receptor (D); {beta}-actin served as a reference gene to normalize RNA concentrations (representative of n = 4). M denotes DNA size ladder.

 
To assess whether the observed changes in Alox15 and ALX mRNA were a specific feature of corneal wounding by complete epithelial removal, we also analyzed expression of both genes after thermal cauterization (see Fig. 7), since it induces a partial 1–2-mm corneal injury. RT-PCR analysis demonstrated that thermal injury induced a similar profile of Alox15 expression as did de-epithelialization, namely a decrease in mRNA expression at 24 h and a return to basal levels by 48–96 h (Fig. 2). By contrast, ALX mRNA expression did not change with thermal injury. By a direct comparison, we also analyzed mRNA expression of 5-LOX, since it is an essential enzyme in the formation of LXA4 with predominant expression in leukocytes. Analyses of uninjured (healthy) corneas from three different strains of mice (BALB/c, C57b/J6, and 129/F2J) demonstrated mRNA expression of Alox5. Corneal injury induced a marked increase in Alox5 mRNA expression 24–96 h after thermal cauterization that was distinct from the expression profile of ALX and Alox15.



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  		FIG. 7.
Anti-inflammatory lipid autacoids limit corneal injury and promote wound healing. Animals were anesthetized, left corneas were cauterized (~1–2 mm) with a heated microprobe, and thermal injury was documented with a CCD camera attached to a slit lamp biomicroscope. After injury, mice were treated by eye drop with the indicated lipid autacoids (1 µg three times daily) or saline alone for 96 h. At the indicated times, wound healing, opacity, and neovascularization were documented and assessed. Digital photographs are representative of 3–4 independent experiments.

 



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  		FIG. 2.
Expression of 12/15-LOX, 5-LOX, and ALX receptor mRNA in normal and thermally injured corneas. Mouse (BALB/c) corneas were collected at the indicated times after thermal injury (see "Experimental Procedures") or from healthy eyes (uninjured, N). Attached noncorneal tissue was carefully removed by dissection, and total RNA was extracted. mRNA expression of selected genes was analyzed by semiqualitative RT-PCR (see "Experimental Procedures") employing specific primers for mouse 12/15-LOX (Alox15), ALX receptor, and actin with PCR amplification of 35 and/or 25 cycles. 5-LOX (Alox5) expression was assessed in healthy and injured corneas of BALB/c mice as well as in uninjured corneas of C57b/J6 and 129/F2J mice. All results are representative of at least three independent experiments.

 
In addition to 12/15-LOX (Alox15), mice express several 12-LOX enzymes, of which platelet-type (Alox12) and epidermal-type 12-LOX (Aloxe) show prominent expression in epithelial cells (45, 48, 51, 55). RT-PCR analysis for Alox15, Alox12, Aloxe, and Alox5 expression demonstrated the mRNA for all genes in the cornea (Supplemental Fig. 1). However, based on semiqualitative mRNA analysis Alox15 was the most abundant transcript in the uninjured cornea.

LXA4 Is a Prominent Endogenous Eicosanoid Generated in Healthy and Injured Corneas—To determine whether LXA4 can be formed in the mouse cornea, we collected healthy corneas and corneas 24 h after thermal injury, since two key enzymes for LXA4 biosynthesis, 5-LOX and 12/15-LOX, demonstrate mRNA expression in this tissue (Figs. 1 and 2). Corneas were exposed to the nonselective agonist calcium ionophore A23187 [GenBank] to amplify activation of pathways for endogenous eicosanoid formation. DAD-RP-HPLC analysis (Fig. 3A, n = 3) demonstrated a peak in both injured and healthy corneas that matched the characteristic chromophore of a conjugated tetraene chromophore with a {lambda}max of 301 nm and coeluted with the synthetic LXA4 standard (Fig. 3A). To assess the endogenous formation of 5-LOX products, a key enzyme of PMNs, we assessed formation of LTB4 in corneas 24 h post-thermal injury (n = 2). DAD-RP-HPLC analysis (Fig. 3B) demonstrated a peak that matched the triene chromophore and retention time of LTB4 in calcium ionophore-activated corneas. In the absence of calcium ionophore stimulation, LTB4 formation was not detected by DAD-RP-HPLC analysis in injured corneas (n = 2). By contrast, corneas 24 h after thermal injury formed endogenous LXA4 without requiring calcium ionophore stimulation as evidenced by the DAD-RP-HPLC analysis (n = 3) shown in Fig. 3C. Furthermore, corneas wounded by de-epithelialization also demonstrated endogenous formation of a peak (Fig. 3D) that matched the characteristic chromophore and retention time of LXA4 (n = 2).



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  		FIG. 3.
HPLC identification of endogenous LXA4 formation in healthy and injured mouse corneas. Healthy (no injury) and injured corneas were gently homogenized in cold MeOH and extracted by ODS-C18 solid phase and methyl formate fractions analyzed by DAD-RP-HPLC. A, corneas (2 per analysis) were collected 24 h after thermal injury or from healthy mouse eyes and immediately placed in buffered saline (37 °C) and incubated in the presence of calcium ionophore (A23187 [GenBank] , 5 µM) for 30 min. Shown is a representative (n = 3) DAD-UV analysis at 301 nm for tetraene-carrying compounds in injured and healthy corneas and of the LXA4 standard (5 ng). The inset shows the UV chromophore of the endogenous compound that matches the retention time and chromophore of synthetic LXA4. B, corneas (4 per analysis) were collected 24 h after thermal injury and either immediately processed for analysis or placed in buffered saline (37 °C) and incubated in the presence of calcium ionophore for 30 min prior to processing. Shown is a representative (n = 2) DAD-UV analysis at 270 nm for triene-carrying compounds in the injured corneas. The inset shows the UV chromophore of the endogenous compound that matches LTB4 standard retention time and chromophore. C, corneas (4 per analysis) were collected 24 h after thermal injury and immediately processed for analysis. Shown is a representative (n = 3) DAD-UV analysis at 301 nm. The inset shows the UV chromophore of the endogenous compound that matches LXA4 retention time and chromophore. D, corneas (3 per analysis) were collected 48 h after complete epithelial debridement and immediately processed. Shown is a representative (n = 2) UV-DAD analysis at 301 nm. The inset shows the UV chromophore of the endogenous compound that matches LXA4 retention time and chromophore. mAU, milliabsorbance units.

 
In order to investigate relative quantitative changes in endogenous LXA4 formation, we took advantage of a commercially available and established LXA4 ELISA (54). To directly assess whether endogenous formation of LXA4 correlates with Alox15 expression and the rate of wound healing, we utilized corneal de-epithelialization as a quantitative and reproducible injury model. In addition to LXA4, we quantitated the formation of PGE2 and LTB4 as markers of COX-2 induction and leukocyte activation, respectively (17, 56, 57). Analyses demonstrated that LXA4 (immunoreactive LXA4; 2.5 ± 0.4 ng/cornea) is a predominant eicosanoid in uninjured corneas (Fig. 4, n = 7) when directly compared with PGE2 (immunoreactive PGE2; 0.9 ± 0.3 ng/cornea) and LTB4 (immunoreactive LTB4 0.2 ± 0.05 ng/cornea), based on the relative immunoreactive levels obtained from the specific ELISAs. Epithelial removal induced a significant (p < 0.001) and sustained increase (24–96 h) in immunoreactive PGE2 (7.0–7.2 ng/cornea, n = 3–4), whereas immunoreactive LTB4 levels did not change (p > 0.05, n = 3–4) despite a significant infiltration of PMN into the stroma (see Fig. 1B). In sharp contrast, immunoreactive LXA4 levels in the injured and healing cornea correlated with the temporal profile of Alox15 expression and re-epithelialization (see Fig. 1, A and C). Loss of the epithelium was associated with a 54% (1.1 ± 0.2 ng/cornea, 6 h, p = 0.0027, n = 4) decrease in immunoreactive LXA4 levels, which increased at 48 h to 3.8 ± 0.4 ng (p = 0.009, n = 11), the peak of PMN infiltration (see Fig. 1B), and returned to base line by 96 h (2.9 ± 0.3 ng/cornea, n = 10). It is important to note that the immunoreactive LXA4 levels measured by ELISA from single de-epithelialized cornea (1.1–3.8 ng) are higher than those detected by direct DAD-RP-HPLC analysis (~0.2–0.5 ng/cornea). Hence, detailed quantitative analysis by physical methods such as LC/MS/MS must be carried out to clearly define the endogenous concentrations of LXA4 in healthy and injured corneas.



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  		FIG. 4.
LXA4 is a prominent endogenous eicosanoid generated in healthy and injured corneas. Healthy (no injury) and de-epithelialized corneas were dissected at the indicated time points and immediately placed in MeOH, extracted, and analyzed by specific ELISAs (see "Experimental Procedures"). A, endogenous LXA4 formation was quantitated in corneas from uninjured eyes (n = 7) or in eyes 6 (n = 4), 24 (n = 11), 48 (n = 11), or 96 h (n = 10) after epithelial removal. *, significant differences (ANOVA, p < 0.03) from uninjured corneas. B, endogenous PGE2 formation in corneas after epithelial removal (n = 3–4). *, significant difference (ANOVA, p < 0.001) from uninjured corneas. C, temporal profile of endogenous LTB4 formation in injured corneas (n = 3–4).

 
Endogenous Formation of NPD1 in the Cornea—We set out to determine whether NPD1, a novel DHA derived 15-LOX product, is formed in corneas of mice that were placed on an {omega}-3 fatty acid-enriched diet. We analyzed formation of endogenous NPD1 in corneas from uninjured eyes ex vivo and in vivo in corneas that were collected 24 h after thermal injury. In uninjured isolated corneas, DAD-RP-HPLC analysis (n = 4) identified the presence of a peak that carried a characteristic chromophore for a conjugated triene double bond structure (Fig. 5A) and coeluted with an NPD1 standard. Moreover, analysis also identified a peak (Fig. 5B) that matched retention time and a characteristic diene chromophore of 17S-HDHA standard. Formation of NPD1 and 17-HDHA was also detected in injured corneas (n = 4). Fig. 5, C and D, shows identification of DHA-derived mediators based on their specific retention time and UV chromophore. It is important to note that endogenous NPD1 and 17S-HDHA formation was not detected by DAD-HPLC analysis in corneas from animals that were placed on a corn oil based control diet (n = 2).



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  		FIG. 5.
Endogenous formation of NPD1 in the cornea. A and B, RP-HPLC-DAD identification of endogenous NPD1 and 17-HDHA formed ex vivo. Corneas were collected from animals that were placed on a low fat {omega}-3 enriched diet and incubated in the presence of a calcium ionophore (A23187 [GenBank] , 5 µM). Reaction products were extracted and analyzed by RP-HPLC (see "Experimental Procedures"). DAD-UV analyses at 270 nm for triene-carrying (A) or at 235 nm for diene-carrying (B) compounds are shown. Retention times of authentic standards are indicated by arrows, and insets show the chromophores from endogenous compounds (*) that match NPD1 and 17S-HDHA. C and D, RP-HPLC-DAD identification of NPD1 and 17-HDHA formation in vivo. Corneas were collected 24 h after thermal injury and immediately processed. Shown are the DAD-UV analyses at 270 nm (C) and 235 nm (D). Retention times of authentic standards are indicated by arrows. Results are representative of n = 3–4.

 
GC/MS Identification of Endogenous NPD1 in the Injured Cornea—We isolated 17-HDHA and NPD1 from injured corneas by RP-HPLC and analyzed the derivatized compounds by GC/MS using selective ion monitoring. The pentafluorobenzyl ester and trimethylsilyl ether derivatives eluted beneath a single peak in their respective GC-selective ion chromatogram (Fig. 6, A and C). Full scanning analysis of the NPD1 material (Fig. 6D) revealed an anion of strong intensity at m/z 503 with two weaker ions at m/z 413 and m/z 323, which is consistent with a dihydroxy-DHA structure (see Fig. 6D, inset), whereas the 17-HDHA material revealed (Fig. 6B) an anion of strong intensity at m/z 416 with a weaker ion at m/z 325, which is consistent with a monohydroxy-DHA structure (see Fig. 6B, inset). Taken together, data shown in Figs. 5 and 6 are consistent with established physical criteria for 17S-HDHA and NPD1 (35–37, 46) and provide strong evidence for their endogenous formation in the cornea.



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  		FIG. 6.
GC/MS identification of endogenous 17-HDHA and NPD1 in injured cornea. Peaks coeluting with authentic standards for 17S-HDHA and 10,17S-diHDHA (NPD1) were isolated by RP-HPLC (see Fig. 3) and derivatized. 10–100 pg were analyzed by GC/MS using a 6890 GC system with a HP5MS cross-linked ME siloxane column (30 m x 0.25 mm x 0.25 µm), an autosampler, and a 5973 mass-selective detector. A, selective ion chromatogram at m/z 325; retention time of authentic 17-HDHA is indicated by an arrow. B, MS spectrum of endogenous 17-HDHA. The inset shows signature ions for a mono-hydroxy-DHA compound. C, selective ion chromatogram at m/z 503; retention time of authentic NPD1 is indicated by an arrow. D, MS spectrum of endogenous NPD1; the inset shows diagnostic ions for a dihydroxy-DHA compound (depicted with tentative double bond geometry). Results are representative of n = 4.

 
Anti-inflammatory Lipid Autacoids Limit Corneal Injury and Promote Wound Healing—A bioaction for LXA4 or NPD1 has not been identified in the cornea; thus, we assessed the impact of these anti-inflammatory lipid autacoids on the sequelae of thermal injury. Cauterization induced a significant wound that was characterized by wound closure at 4–7 days, neovascularization at 4 days, and corneal opacity (Fig. 7, n = 3–4). Topical treatment (1 µg/cornea three times daily) with LXA4 or 17S-HDHA, the metabolic precursor to NPD1, demonstrated a consistent (n = 3–4) and pronounced acceleration in wound closure at 24 h, which was followed by attenuation of corneal opacity and neovascularization by 96 h (Fig. 7) when directly compared with animals that received saline treatment alone (n = 4).

Anti-inflammatory Lipid Autacoids Promote Re-epithelialization—In view of the impact of 17S-HDHA and LXA4 treatment on wound closure at 24 h (see Fig. 7), which preceded the subsequent attenuation of neovascularization and corneal opacity, we hypothesized that these anti-inflammatory lipid autacoids exhibit a novel bioaction in epithelial wound healing. To test this hypothesis, we removed the epithelium while leaving the underlying stroma intact and quantitated re-epithelialization (Fig. 8). Treatment with LXA4, 17S-HDHA, and NPD1 significantly increased re-epithelialization of denuded corneas at 24 h (Fig. 8B, p < 0.003) by 72 ± 23% (n = 10), 114 ± 22% (n = 4), and 90 ± 8% (n = 5), respectively, when directly compared with saline-treated mice (n = 10). A similar pronounced increase in the rate of re-epithelialization (Fig. 8, A and C, p < 0.001), following treatment with the anti-inflammatory mediators, was observed at 48 h, namely 69 ± 8% (LXA4, n = 12), 76 ± 12% (17S-HDHA, n = 6), and 68 ± 12% (NPD1, n = 5). Similar results were obtained with a methyl ester LXA4 analog, ATLa, at a 1-µg topical dose (increased re-epithelialization with ATLa: 72 ± 15% at 24 h (n = 8) and 70 ± 17% at 48 h (n = 6)) that is a well documented topically active anti-inflammatory lipid mediator (58–60). By contrast, treatment with the proinflammatory eicosanoids, LTB4 or 12R-HETrE, at a dose of 1 µg/treatment (n = 5–6), did not impact epithelial wound closure (Fig. 8, B and C, p > 0.05). These findings identify a novel bioaction for LXA4 and NPD1 in epithelial wound healing.



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  		FIG. 8.
Anti-inflammatory lipid autacoids promote re-epithelialization. A, digital photograph of healing corneas. The corneal epithelium was removed by an Algerbrush II corneal rust ring remover, and eyes were treated topically (1 µg three times daily) with the indicated lipid autacoids or saline alone. 48 h after epithelial removal, animals were anesthetized, and the denuded area of corneas was stained with fluorescein. Shown are images from three representative independent experiments at 16x magnification (n = 8–14). Quantitation of wound closure (re-epithelialization) 24 h (B) and 48 h (C) after corneal de-epithelialization is shown. Mice were treated topically by eye drops (1 µg three times daily) with LXA4 (n = 10–12), 17S-HDHA (n = 4–6), NPD1 (n = 5), LTB4 (n = 5–6), or 12R-HETrE (n = 5–6), and re-epithelialization/wound closure is expressed as the percentage change from saline-treated eyes (24 h: 11.3 ± 1.9%, n = 12; 48 h: 40.6 ± 3.1%, n = 10). *, significant difference from saline treatment alone (ANOVA, p < 0.001).

 
Anti-inflammatory Lipid Autacoids Inhibit Chemokine Formation but Do Not Impair PMN Recruitment to Injured Corneas—We treated corneas after epithelial removal with anti-inflammatory lipid mediators to assess their action on the injury-induced infiltration of PMN (see Fig. 1B). Topical treatment (1 µg three times daily/cornea) with either LXA4 (n = 3–8), 17S-HDHA (n = 3–5), or NPD1 (n = 4–5) did not inhibit PMN recruitment to the cornea at 24 or 48 h after injury (Fig. 9) but, to the contrary, demonstrated a significant increase in PMN at 48 h (Fig. 9B, p < 0.03) with LXA4 (61,342 ± 14,189 PMN/cornea), 17S-HDHA (59,723 ± 7039 PMN/cornea), and NPD1 (62,541 ± 8729 PMN/cornea) treatment when directly compared with corneas from saline-treated mice (33,097 ± 3948 PMN/cornea, n = 6–8). Similar results were obtained with the methyl ester LXA4 analog (ATLa, 60,120 ± 11,791 PMN/cornea, n = 6), which is a well documented topically active anti-inflammatory lipid mediator (58–60). In direct comparison, 12R-HETrE also induced a significant (n = 4, p < 0.04) increase in PMN at both 24 h (41,506 ± 11,359 PMN/cornea) and 48 h (65,035 ± 7982 PMN/cornea), whereas topical treatment with LTB4 showed a trend of increased PMN infiltration at 48 h (61,317 ± 24,884 PMN/cornea) that was not significant (p = 0.078). These results are consistent with MPO data obtained in the thermal injury model (n = 3) that demonstrate that neither treatment with LXA4 (110,384 ± 21,560 PMN/cornea) nor 17S-HDHA (161,279 ± 25,193 PMN/cornea) inhibited PMN recruitment to injured corneas (saline treatment alone = 115,617 ± 23,384 PMN/cornea). In sharp contrast, topical treatment with 17S-HDHA, NPD1, and the analog of LXA4 (ATLa) markedly reduced formation (~60%) of the proinflammatory chemokine KC (Fig. 9, C and D), the mouse homolog to human IL-8, a prominent chemokine in acute inflammatory lesion, keratitis, and an angiogenic factor in the cornea (61–64). Therefore, the current results provide evidence that the topical action of LXA4 and NPD1 on re-epithelialization (Fig. 8) is not due to inhibition of PMN recruitment to injured corneas and identify a novel epithelial targeted bioaction for these lipid autacoids.



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  		FIG. 9.
Anti-inflammatory lipid autacoids inhibit chemokine formation but do not impair PMN recruitment to the injured cornea. Corneas were collected from injured eyes 24 and 48 h after removal of the epithelium. Following epithelial removal (Fig. 6), mice were treated topically by eye drops (1 µg three times daily) with the indicated lipid autacoids (n = 4–8) or saline alone (n = 6–8) for 24 (A) or 48 h (B). Corneas were dissected from injured eyes under a microscope, and MPO activities were measured as a quantitative marker of PMN. *, significant differences from saline treatment alone (ANOVA, p < 0.01). C, corneas were dissected 24 and 48 h after de-epithelialization or from healthy (uninjured) eyes. Corneas were pooled (2 mice per data point) and homogenized in phosphate-buffered saline on ice in the presence of protease inhibitors. KC formation (mouse IL-8 homolog) was quantitated using a specific custom sandwich ELISA (SearchLight Protein Arrays). D, eyes were treated topically with either 17-HDHA, NPD1, or a stable LXA4 analog, ATLa (1 µg three times daily) as indicated. Corneas were collected 48 h after de-epithelialization and homogenized, and KC formation was analyzed by ELISA. Data are expressed as percentage change from the saline-treated control (C). All samples for KC determination were analyzed in duplicate, and shown are the average from two independent experiments (4 mice/data point).

 
12/15-LOX-deficient Mice Exhibit a Phenotype of Delayed Wound Healing, Impaired LXA4 Biosynthesis, and Attenuated PMN Recruitment—We utilized the well characterized Alox15-deficient mice (12/15-LOX-/-) to confirm a role for this path-way in epithelial wound healing. When directly compared with matched congenic controls (12/15-LOX+/+), 12/15-LOX-/- mice showed a significant 65 ± 5% (p = 0.0009, n = 4) and 38 ± 4% (p = 0.04, n = 4) decrease in re-epithelialization 24 and 48 h, respectively, after epithelial removal (Fig. 10, A, B, and E). Delayed epithelial wound healing was associated with a significant decrease (43%, p < 0.05; Fig. 10C) in endogenous LXA4 formation (immunoreactive LXA4 4.1 ± 1.0 ng/cornea, 12/15-LOX+/+ mice; immunoreactive LXA4 2.3 ± 0.7 ng/cornea, 12/15-LOX-/- mice) as well as a decrease (p = 0.013) in infiltrating PMN (Fig. 10D) by 47 ± 5% in 12/15-LOX-/- mice (13,727 ± 2125 PMN/cornea, n = 3) when directly compared with their congenic controls (26,600 ± 3066 PMN/cornea, n = 3). These results identify a new phenotype for 12/15-LOX-/--deficient mice, namely delayed epithelial wound healing that showed a correlation with impaired endogenous LXA4 formation and recruitment of PMN to the injured cornea.



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  		FIG. 10.
12/15-LOX-deficient mice exhibit a phenotype of delayed wound healing, impaired LXA4 formation, and attenuated PMN recruitment. Animals were anesthetized, and corneal epithelium was removed from 12/15-LOX (Alox15)-deficient mice (12/15-LOX-/-, n = 4) and their congenic matched controls (12/15-LOX+/+, n = 4). At 24 h (A) and 48 h (B), corneas were stained with fluorescein, and re-epithelialization was quantitated by image analysis. Re-epithelialization is expressed as the percentage decrease in the denuded corneal area. C, 48 h post-de-epithelialization corneas from 12/15-LOX-/- and 12/15-LOX+/+ mice (n = 3) were collected and immediately processed (see "Experimental Procedures") for analysis by a specific LXA4 ELISA. D, corneas were dissected at 48 h post-de-epithelialization, and MPO activity was measured (see "Experimental Procedures") as a quantitative marker of PMN content (n = 3). *, significant difference (p < 0.04) from the congenic controls. E, representative (n = 4) digital image of fluorescein-stained eyes 48 h after epithelial removal at x16 magnification.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented identify endogenous formation of the eicosanoid LXA4 and DHA-derived NPD1 in healthy and injured corneas. In addition, results demonstrate predominantly constitutive expression of 12/15-LOX (Alox15) and the LXA4 receptor mRNA in epithelial cells of healthy corneas as well as pronounced changes in 12/15-LOX mRNA expression after corneal injury, which correlates with epithelial wound healing. These lipid autacoids and their biosynthetic and signaling pathways are key components of an important endogenous anti-inflammatory and immunoregulatory pathway that promotes resolution of inflammation (3, 21, 24, 25, 27, 35, 37). Defining characteristics of the cornea are immune privilege and suppression of immunogenic inflammation, whose mechanism remains to be clearly defined; thus, identification of an endogenous anti-inflammatory pathway in this tissue is of interest. Amplification of this pathway by topical application of LXA4 or NPD1 following corneal injury markedly accelerated epithelial wound repair, abrogated chemokine formation (KC), and, subsequently, limited the sequelae of corneal injury without impairing PMN recruitment. Moreover, Alox15-deficient mice (12/15-LOX-/-) exhibit a phenotype of delayed wound healing and attenuated PMN recruitment that correlated with impaired LXA4 formation. Hence, the present findings provide evidence for a novel role of 12/15-LOX pathways and its products, LXA4 and NPD1, in wound healing that is distinct from their well established action of attenuating PMN trafficking.

Effective host defense requires initiation of an acute inflammatory response that ultimately leads to removal of the foreign challenge and restoration of tissue structure and function. It has become evident that the epithelium in mucosal tissues plays a key role in coordinating host responses to infection or injury, not only by providing a critical barrier but also by generating bactericidal peptides, proinflammatory chemokines such as IL-8, and by presenting antigens (52, 65, 66). However, the role of the corneal epithelium in coordinating inflammation and immune responses remains to be delineated. Here we provide, to our knowledge, the first evidence for a potential role of the epithelium in modulating inflammatory and immune responses in the cornea, namely as a cell type that initiates formation of LXA4 and NPD1. This conclusion is supported by evidence shown in Figs. 1, 2, 3, 4, which demonstrates that mRNA expression of Alox15 and endogenous formation of LXA4 are abrogated by loss of the epithelium and restored during epithelial wound healing. It is important to note that two independent methods, ELISA and DAD-RP-HPLC analysis, confirmed endogenous LXA4 formation, and despite a difference in the absolute quantitative levels, both methods suggest epithelium-dependent formation of LXA4 in the cornea.

Formation of both LXA4 and NPD1 correlated with mRNA expression of leukocyte-type 12/15-LOX (Alox15), the well characterized mouse homolog of the human 15-LOX type 1 (ALOX15). 15-LOX (Alox15, ALOX15) is expressed in leukocytes and epithelial cells in both humans and mice; thus, we focused our analysis on this member of the mouse 12-LOX enzymes since semiqualitative RT-PCR analysis demonstrated that Alox15 is an abundant transcript in the mouse cornea (Figs. 1 and 2 and Supplemental Fig. 1). Furthermore, evidence from Western immunoblot analysis indicates that platelet-type 12-LOX (Alox12) protein is not expressed in the eye (48), and studies with recombinant enzyme demonstrate that DHA is an inefficient substrate for the epidermal-type 12-LOX (Aloxe) (55). However, although prominent 15-LOX (ALOX15, ALOX15B) expression and activity has been identified in human corneal epithelial cells and our data suggest that 12/15-LOX (Alox15) is an abundant mRNA transcript in the mouse cornea, we cannot exclude a role for other mouse 12-LOX enzymes, platelet-type 12-LOX (Alox12), epidermal-type 12-LOX (Aloxe), and epidermis-type 12-LOX (Aloxe3), in the formation of LXA4 and NPD1.

Epithelial removal and thermal injury were associated with temporally defined PMN infiltration, so it is likely that PMN contributes to NPD1 or LXA4 formation in the wounded cornea, especially since the 5-LOX pathway in PMN is an essential enzyme for LXA4 formation during heterotypic cell interactions at sites of acute inflammation (18, 37). However, two major findings negate PMN as an essential cell type for LXA4 and NPD1 formation in the cornea: 1) both LXA4 and NPD1 were formed, as evidenced by ELISA, HPLC, and/or GC/MS analysis, by the uninjured corneas which contained no PMN; and 2) formation of LXA4 and Alox15 expression after epithelial removal was abrogated at 24 h, coinciding with a significant increase in PMN, and restored to basal levels at 96 h despite a decrease in corneal PMN. Moreover, 5-LOX mRNA expression remained markedly elevated despite a significant decrease in corneal PMN, 96 h after corneal injury, which is consistent with the prolonged presence of other 5-LOX-expressing leukocytes such as macrophages in injured corneas. 5-LOX expression or activity has been demonstrated in fibroblasts, epithelial cells, and antigen-presenting cells, all of which hold a key role in the inflammatory/reparative response of the cornea. Hence, it remains to be investigated to what extent these cell types contribute to 5-LOX activity in the healthy cornea and how corneal injury impacts expression and 5-LOX activity in resident and infiltrating cells.

It is striking that formation of LTB4, a major metabolite of the PMN 5-LOX pathway, did not change after corneal injury despite pronounced PMN infiltration at 24 and 48 h. The observation that endogenous LTB4 formation did not correlate with transient PMN infiltration is consistent with the notion that the degree of PMN activation is dependent on the type of corneal injury and that surgical procedures such as keratectomy are generally not associated with PMN-mediated injury. The mediators that suppress full PMN activation and inhibit LTB4 formation in the immune privileged cornea remain to be identified. However, it is of interest that several reports have demonstrated that LXA4, an endogenous eicosanoid in the healthy and injured mouse corneas (Figs. 3 and 4), inhibits PMN activation in several in vitro and in vivo models of acute inflammation (18) and that its metabolic precursor 15-HETE, a product of the 15-LOX pathway, inhibits LTB4 formation in PMN (67, 68). Hence, it is not unreasonable to speculate that LXA4 formation in the healthy and injured corneas provides a mechanism to protect against PMN-mediated tissue injury.

The potential clinical relevance of NPD1 and LXA4 formation in injured corneas is highlighted by their impact on the sequelae of corneal injury. Therapeutic amplification of LXA4 and NPD1 by direct topical application resulted in a pronounced acceleration of wound closure as well as inhibition of the chemokine KC, the mouse homolog to IL-8, which has been implicated as a key mediator of acute inflammation and angiogenesis in the eye (61–64). Unexpectedly, this beneficial action was not due to inhibition of leukocyte recruitment to the injured cornea, since treatment with anti-inflammatory lipid autacoids actually enhanced PMN content of the cornea by 48 h. The role of PMN in the reparative response is not clearly defined and is probably tissue-dependent, but early studies indicate no significant impact on wound healing (11). In contrast, in vivo studies in the eye demonstrate that complete inhibition of PMN infiltration into the cornea can impair wound healing (12). By direct comparison, 12R-HETrE, a proinflammatory eicosanoid with well defined bioactions in the cornea (14), also increased PMN infiltration and, like LTB4, a potent PMN activator, did not promote re-epithelialization of denuded corneas. These findings suggest that an increase in PMN infiltration and/or activation alone is not sufficient to promote wound healing. Furthermore, they identify a novel epithelial targeted action for topical LXA4 and NPD1 in the avascular cornea.

Direct evidence for a key role of the 12/15-LOX pathway in epithelial wound healing was obtained from experiments with Alox15-deficient mice (12/15-LOX-/-). Our findings identify a previously unknown phenotype of delayed wound healing in these mice. Moreover, the delayed wound healing was associated with a pronounced decrease in PMN in the injured cornea and impaired endogenous LXA4 formation in 12/15-LOX-/- mice. These findings are consistent with the topical action of exogenous LXA4 in the injured cornea, which accelerated re-epithelialization but was also associated with increased PMN recruitment. Hence, targeted deletion of 12/15-LOX in vivo uncovered a key role for this pathway and its metabolites in epithelial wound healing. In this regard, it is noteworthy that epidermal wounding experiments in platelet-type 12-LOX (Alox12)-deficient mice indicate that this mouse 12-LOX enzyme, unlike 12/15-LOX (Alox15), does not impact dermal wound healing or inflammation (45, 69). However, it is likely that other mouse LOX pathways contribute to endogenous LXA4 formation in the eye, since 12/15-LOX-/- mice, despite a significant decrease in endogenous formation, were still able to generate LXA4. Inhibition of PMN trafficking to a site of acute inflammation is a well established bioaction of LXA4, LXA4 stable analogs, and NPD1 (25, 27, 35, 37). Thus, it is important to note that the avascular cornea exhibits atypical inflammatory and immune responses and that topical treatment with anti-inflammatory lipid mediators accelerated wound healing and limited corneal injury, despite an increase in PMN. Taken together, these results suggest that PMN in the presence of an anti-inflammatory pathway does not impair epithelial wound healing. Therefore, in this context, amplified recruitment of PMN to the de-epithelialized cornea has to be viewed as a beneficial protective response against potential infection.

Treatment with anti-inflammatory lipid mediators markedly accelerated epithelial wound closure, which preceded attenuation of neovascularization and corneal opacity. These results strongly indicate an epithelial directed action of LXA4 and NPD1, and evidence for this conclusion is 3-fold: 1) LXA4 and NPD1 accelerate re-epithelialization (Fig. 8); 2) LXA4 and NPD1 inhibit KC formation, a product of epithelial cells and fibroblasts (Fig. 9, C and D); and 3) corneal epithelial cells express the LXA4 receptor (Figs. 1 and 2). Several studies support an epithelial directed bioaction of LXA4, since they have demonstrated that ALX mediates inhibition of pathogen-induced IL-8 formation (52, 70) and stimulates host defense by inducing expression of bactericidal/permeability-increasing protein in colonic epithelial cells (65). Moreover, a recent report demonstrates that NPD1 inhibits apoptosis in human retinal pigmented epithelial cells (36). The cellular mechanism by which LXA4 and NPD1 exert their effects on wound healing need to be explored, but our data indicate that it may include receptor-mediated action on epithelial cell proliferation. In this regard, it is of interest that the 15-LOX (ALOX15) pathway in humans has been implicated in several forms of epithelial cell cancers as a component of the cell proliferation process (49, 71) and that expression of the ALX receptor gene has been identified in human corneas and epithelial cells (52).

This report also provides the first evidence (Figs. 5 and 6) that a {omega}-3-enriched diet can induce endogenous formation of DHA-derived NPD1 and 17S-HDHA and is the first identification of these anti-inflammatory lipid autacoids in the cornea. These findings are consistent with endogenous formation of NPD1 in both mouse brain and human retinal pigmented epithelial cells via a 15-LOX-like mechanism (35–37). The observation that a {omega}-3 diet can induce formation of an additional anti-inflammatory signal may provide a molecular mechanism for several reports that have demonstrated a beneficial action of both dietary and topical {omega}-3 fatty acids in reducing inflammation in immunogenic keratitis (32, 33). However, additional long term dietary studies are required to clearly define the impact of dietary {omega}-3 fatty acids on the profile of lipid autacoids and the inflammatory/reparative process.

In summary, the present study identifies a novel role for both the mouse 12/15-LOX pathway and LXA4 and NPD1 in wound healing that is distinct from their PMN-directed bioactions and adds evidence for their role as important signals that promote resolution of the inflammatory/reparative response. These findings are potentially clinically relevant, since they identify endogenous signals that are topically active and exhibit multi-pronged protective properties that limit inflammation while promoting host defense and wound healing.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK6053 (to K. G.) and EY06513 (to M. L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains an additional figure. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology, NY Medical College, Valhalla, NY 10595. Tel.: 914-594-4625; Fax: 914-594-4273; E-mail: karsten_gronert{at}nymc.edu.

1 The abbreviations used are: PMN, polymorphonuclear leukocyte(s); DHA, docosahexaenoic acid; NPD1, neuroprotectin D1 (10,17S-diH-DHA); diHDHA, dihydroxy-DHA; LXA4, lipoxin A4; LTB4, leukotriene B4; PGE2, prostaglandin E2; 12R-HETrE, 12R-hydroxy-eicosatrienoic acid; GC, gas chromatography; MS, mass spectrometry; DAD, diode array detector; RP, reverse phase; HPLC, high performance liquid chromatography; MPO, myeloperoxidase; LOX, lipoxygenase; ALX, LXA4 receptor; HBSS, Hanks' balanced salt solution; ATLa, 15-epi-16-(para-fluorophenoxylipoxin A4-methyl ester; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance; LC, liquid chromatography; IL, interleukin; S, sinister; R, rectus. Back


   ACKNOWLEDGMENTS
 
We thank Steven Tresker and Daniel DiLeo for technical assistance, Rowena Kemp for expert assistance in the GC/MS analysis, and Melody Steinberg for editing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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