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Published as doi: 10.1096/fj.06-7918com.
 (The FASEB Journal. 2007;21:2257-2266.)
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Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing

Benjamin Biteman, Iram R. Hassan, Everald Walker, Alexander J. Leedom, Michael Dunn, Francesca Seta, Michal Laniado-Schwartzman and Karsten Gronert1

Department of Pharmacology, New York Medical College, Valhalla, New York, USA

1Correspondence: Department of Pharmacology, New York Medical College, Basic Science Bldg., Valhalla, NY 10595, USA. E-mail: karsten_gronert{at}nymc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the immune-privileged cornea, epithelial wounds heal rapidly with almost no scarring and, unlike in most other tissues, acute inflammation in the absence of infection is beneficial to healing. Molecular mechanisms, which account for this striking property, remain to be clearly defined, but they likely include autacoids that control leukocyte activation. Two prominent enzymes, 12/15-lipoxygenase (LOX), which generates antiinflammatory lipid autacoids, and heme-oxygenase (HO), which generates antioxidants and carbon monoxide, are highly expressed in human and mouse corneas. LXA4, an endogenous 12/15-LOX product, proved to be a potent inhibitor of exacerbated inflammation and significantly increased re-epithelialization in corneal wounds. In vivo deletion of 12/15-LOX correlated with exacerbated inflammation and impaired wound healing in 12/15-LOX–/– mice, a phenotype that was rescued by treatment with LXA4. More importantly, 12/15-LOX–/– mice demonstrated impaired induction of HO-1 in both acute and exacerbated inflammation. Topical LXA4 restored HO-1 expression in 12/15-LOX–/– mice and amplified HO-1 gene expression in human corneal epithelial cells. HO-2–/– mice, which fail to induce HO-1, also demonstrated exacerbated inflammation in response to injury, a phenotype that, notably, correlated with a 50% reduction in endogenous LXA4 formation. Collectively, results demonstrate a critical role for LXA4 in inflammatory/reparative responses and provide the first evidence that 12/15-LOX and HO systems function in concert to control inflammation.—Biteman, B., Hassan, I. R., Walker, E., Leedom, A. J., Dunn, M., Seta, F., Laniado-Schwartzman, M., Gronert, K. Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing.


Key Words: lipid autacoids • resolution • neutrophil • 12/15-lipoxygenase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INFLAMMATION PLAYS A CRITICAL ROLE in the response to physical injury and is intimately linked to the reparative response. Indeed, execution of an acute inflammatory response and tissue repair depends on the precarious activation of resident cells and recruitment of leukocytes, which is initiated by the rapid formation of small molecules of which arachidonic acid-derived autacoids are some of the earliest signals triggered by injury (1 , 2) . Despite the essential role of leukocytes in maintaining host defense at the site of tissue injury and the critical role of monocytes in tissue remodeling, excessive and chronic inflammation leads to unabated leukocyte infiltration, amplified cytokine and chemokine production and, ultimately, impaired wound healing (1 2 3 4) . In this regard, endogenous pathways that promote the execution of an acute self-resolving inflammatory response while counter-regulating proinflammatory signals are of primary interest.

The challenge of maintaining adequate host defense while actively suppressing inflammatory and immunogenic responses, especially in response to injury, is nowhere as acute and evolutionarily conserved as in the avascular and transparent tissues of the eye, where every inflammatory/reparative response could dramatically impair vision (5 , 6) . Hence, the cornea maintains an immune-privileged and antiinflammatory environment where moderate injury can induce a beneficial and regulated inflammatory response that promotes wound healing (7 8 9 10) . Mechanisms that account for the atypical inflammatory/reparative response are just beginning to emerge, and recent reports suggest that it may depend on the basal level of antiinflammatory signals in the cornea (7 , 11) .

Two distinct and prominent antiinflammatory pathways have been identified in the mouse cornea (7 , 11 12 13 14) , namely 12/15-lipoxygenase (Alox15) and heme-oxygenase (HO); their role in limiting the sequelae of corneal injury has been strongly supported by recent loss of function studies using HO-2 (11) or Alox15 (7) knockout mice. 12/15-LOX is highly expressed in mucosal epithelial cells and is one of the most prominent inducible genes in human monocytes (15) . This important enzyme has a well documented role in regulating physiological and pathophysiological inflammatory/immune functions (15 16 17 18 19 20) . In particular, its enzymatic products, namely lipoxin A4 (LXA4) and its metabolic precursor 15-HETE, are antiinflammatory lipid autacoids that inhibit PMN and lymphocyte activation and dendritic cell function, and promote clearance of apoptotic PMN by macrophages. Hence, this pathway is considered critical to the resolution of acute inflammation (2 , 15 , 19 , 21 22 23 24) . Moreover, 12/15-LOX is a key enzyme in the formation of novel docosahexaenoic acid (DHA)-derived autacoids, namely, 17S,10R-dihydroxy-DHA (25) and 17S-hydroperoxy-DHA, which is a metabolic precursor for the 17S-series resolvins (24) . Recent reports provide compelling evidence that these endogenous DHA-derived autacoids may provide a molecular mechanism for the striking immuno- and neuro-protective actions of DHA (26 27 28 29) .

Heme-oxygenase (HO) exists as two isoenzymes, which are the rate-limiting enzymes in the degradation of cytosolic heme or extracellular heme and is released as a consequence of hemolysis (30 31 32) . HO-2 is believed to function as the constitutive HO activity contributing to cell homeostasis (30 , 32) , and we recently demonstrated that genetic deletion of this basal HO activity impairs induction of the stress gene HO-1 in the cornea (11) . HO-1 has emerged as an inducible protective gene whose amplification by genetic or pharmacological approaches is antiproliferative, antiinflammatory, and antiapoptotic (31 , 33 34 35) . Indeed, important endogenous regulators of the immune response, such as interleukin-10, mediate their protective actions through the activation of the HO-1 system. This fundamental protective circuit is induced in response to oxidative stress and potent activators of inflammatory/immune response such as bacterial lipopolysaccharides. More importantly, recent in vitro studies demonstrate that a stable analog of the aspirin-triggered LXA4, a COX-2 and cytochrome P450-derived epimer of LXA4, induces HO-1 in human endothelial cells (36) . Thus, it is of particular interest that impaired expression of HO-1 manifests itself in a phenotype of exacerbated inflammation and failure to resolve leukocyte infiltration (11) , both of which are key targets for the bioaction of LXA4. However, an interaction between HO and 12/15-LOX systems has not been investigated, especially in the setting of an acute inflammatory/reparative response.

Hence, we set out to define the role of the 12/15-LOX system in an acute inflammatory/reparative response and to elucidate a potential interaction with the cytoprotective HO systems. Here, we report that 12/15-LOX is critical for counterbalancing exacerbated inflammation and to maintain normal wound healing. More importantly, endogenous formation of LXA4 and induction of HO-1 depend in part on the presence of both HO and 12/15-LOX in the injured tissue, which provides the first evidence for a potential amplification loop that may be critical to control the precarious activation of leukocytes and maintain normal wound healing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Balb/c mice (6–8 wk, males) were purchased from Taconic (Bar Harbor, ME, USA). 12/15-lipoxygenase (Alox15) deficient mice (6–10 wk, female) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). This Jax®Gemm® Strain has a targeted mutation in the 15-LOX (Alox15tm1Fun) that is in a background C57BL/6J inbred strain. These mice are well characterized (17 , 20 , 37 , 38) and do not express the "leukocyte-type" 12/15-LOX (Alox15). Age- and gender-matched congenic C57BL/6J stock 000664 mice (6–10 wk, female) were used as controls. Heme-oxygenase-2 null mice (HO-2–/–) are direct descendents of the HO-2 mutants produced by Poss et al. (39) . These well-characterized HO-2 null mice have a C57BL/6 x 129/Sv genetic background (40) , which was used for the age- and gender-matched controls.

Keratitis and epithelial wound healing
All animal studies have been approved by the New York Medical College Vertebrate Animal Committee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Mice were maintained on a 12 h day/night cycle and fed ad libitum a standard diet (Laboratory Rodent Diet #5001, LabDiet, Richmond, IN, USA). 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 corneal injuries. 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 a stereomicroscope as described previously (7) . Corneas were stained with fluorescein (1 mg/ml, Bausch and Lomb, Rochester, NY, USA) as a direct marker of the epithelial defect and the total area of the denuded cornea was quantified by image software analyses. Mice were treated topically (1 eye drop, 5–10 µl) immediately after de-epithelialization with sterile saline or 1–1000 ng (t.i.d.) of indicated lipid autacoids in 100% sterile HBSS (pH 7.4) and/or lipopolysaccharides (LPS, 10 µg, Pseudomonas aeruginosa serotype 10; Sigma, St. Louis, MO, USA). Topical treatments were repeated three times daily (t.i.d.) for 24–96 h. 17S-HDHA (17S-hydroxy-docosa-4Z, 7Z, 11Z, 13Z, 15E, 19Z- hexaenoic acid) and neuroprotectin D1 [10R,17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid, NPD1 (25) ] were generated by biogenic synthesis and purified by HPLC as described previously (41 , 42) ; LXA4 (5S,6R,15S-trihydroxy-eicosa-7E, 9E, 11Z, 13E-tetraenoic acid) and 15S-HETE (15S-hydroxy-eicosa-5Z, 8Z, 11Z, 13E-tetraenoic acid) were purchased from Calbiochem (San Diego, CA, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively.

Assessment of inflammation and wound healing
Eyes of anesthetized mice were stained with fluorescein, and images of the anterior surface taken with a CCD camera were attached to a stereomicroscope to quantify wound closure and re-epithelialization (7) . Digital images were analyzed, and the fluorescein-stained wound area was quantified using image software analysis (Adobe Photoshop, San Jose, CA, USA). For histology, corneas were embedded in paraffin (Paraplast, BDH), sectioned at 5 µm thickness, and stained with hematoxylin-eosin, as described previously (11) . PMN were quantified in dissected and cleaned corneas at indicated time points by measuring myeloperoxidase activity (MPO) as a specific PMN marker, as described previously (7) . In brief, tissues were homogenized in potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide, followed by 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 oxidation 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 peritoneal exudates in Balb/C mice. To quantify chemokines and cytokines as markers of inflammation, corneas (2 corneas per sample) were isolated, cleaned, and homogenized on ice in tissue protein extraction reagent (TPER, Pierce Bioscience, Rockford, IL, USA) containing protease inhibitors (1 protease inhibitor cocktail tablet/10 ml TPER, CompleteMini, Roche Diagnostics, Mannheim, Germany). Cell debris was removed by centrifugation and supernatants were analyzed by Pierce Biotechnology (Woburn, MA, USA) using a custom mouse SearchLight quantitative multiplexed sandwich ELISA Proteome Array.

Cell culture
Human corneal epithelial cells (HCE) were a kind gift from Dr. Haydee Bazan (Louisiana State University, Health Science Center, New Orleans, LA, USA). HCE were grown in complete KBM-2 media (Bullet Kit, Cambrex, Walkersville, MD, USA) to 50% confluency. Complete media was replaced with KBM-2 media containing no growth factors and after 12 h of starvation HCE were treated with 1–1000 nM LXA4, 17S-hydroxy-DHA, NPD1 or KBM-2 media (50 µl) containing <0.001% vehicle (EtOH).

Heme-oxygenase gene and protein expression
RNA from human corneal epithelial cells was isolated using a Absolutely RNA® kit (Stratagene, La Jolla, CA, USA), and RNA integrity was verified by agarose gel electrophoresis and quantified by spectrophotometry. RNA was reverse-transcribed using a Superscript III First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). HO-1 was amplified using 5'-GTCTTCGCCCCTGTCTACTTC-3' and 5'-CTGGGCAAT-CTTTTTGAGCAC-3', ALOX15 5'-CGCTGCGGCTCTGGGA-AATC-3' and 5'-TGTTGGCCGGTGCAGGTGAAGA-3', ALOX15B 5'-TGCCTCTCGCCATCCAGCT-3' and 5'-TGATGGAAGGAG-AACTCGGCAT-3', and ALX (fPRL-1) 5'-CACGGCCACATTACCATTCCT-3' and 5'-AGCGGTCCAGTGCAATGAAA-3'. HO-1 nucleotide primers as well as specific primers for mouse and human reference genes (ubiquitin C, GAPDH, ß-actin) were selected from the Harvard Primer Bank (pga.mgh.harvard.edu/primerbank/) and verified in the NIH Genbank®. Real-time PCR was performed using Brilliant SYBR Green QPCR Master Mix (Stratagene) and an MX3000 QPCR System (Stratagene). All amplifications were run in duplicates, and amplification efficiencies for each primer pair were established. Comparative quantitation of gene expression was determined by MXPro analytical software (Stratagene).

For Western immuno-blot analyses, dissected corneas were homogenized using TPER (Pierce Bioscience) containing protease inhibitors (1 protease inhibitor cocktail tablet/10 ml TPER, CompleteMini, Roche Diagnostic). Homogenate supernatants were concentrated by spin column filtration (Microcon YM-10, Millipore). Protein concentrations were determined by Bio-Rad Bradford Reagent (Hercules, CA, USA). Proteins (5–10 µg) were separated by SDS-PAGE and transferred to nitrocellulose membrane using a semidry transfer apparatus. Membranes were blocked in 7% milk solution and incubated with anti-HO-1 (Stressgen, San Diego, CA, USA) or anti-ß-actin (Sigma) antibody in a 1% milk solution. The membranes were washed and incubated with peroxidase labeled goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Chemiluminescence detection was performed with KPL LumiGLO® kit according to the manufacturer’s instructions.

Endogenous LXA4 formation
Corneas from HO-2–/– and matched WT controls were dissected in cold HBSS buffer without calcium or magnesium and cleaned under a dissecting microscope to remove all noncorneal tissue. Corneas were gently homogenized in 66% MeOH at 4°C and extracted by solid phase, as previously detailed (7 , 43) . To quantify endogenous LXA4 formation, extracted samples (100 µl MeOH) were taken to dryness under a gentle stream of nitrogen and resuspended in ELISA buffer and immediately analyzed by a specific ELISA for LXA4 (44) (Neogen, Lexington, KY, USA) according to manufacturer’s instructions.

Statistical analysis
Student’s t test was used to evaluate the significance of differences between groups, and multiple comparisons were performed by regression analysis and one-way ANOVA (STATISTICA©, StatSoft, Inc., Tulsa, OK, USA). P-values less than 0.05 were considered significant. All data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Epithelial abrasion in the cornea is an established model (7 , 45 , 46) of a self-resolving inflammatory/reparative response that is defined by mild inflammation and complete wound healing within 5–7 d. Endotoxin (LPS) from Pseudomonas aeruginosa, a clinical relevant pathogen associated with bacterial keratitis (47 48 49) , was used as a potent agonist to markedly amplify and sustain inflammation during epithelial wound healing. De-epithelialization induced a consistent corneal wound that exhibited a linear rate of re-epithelialization (Fig. 1 B) and was associated with significant and transient PMN infiltration (Fig. 1A ), consistent with previous reports (7 , 11) . Topical treatment with LPS (10 µg/t.i.d.) markedly amplified PMN content in the cornea by ~3-, 4-, and 2-fold at 24, 48, and 96 h, respectively (Fig. 1A , P<0.01, n=3–11), which was associated with a significant decrease in the rate of epithelial wound healing 47 ± 8% at 24 h, 40 ± 11% at 48 h and 27 ± 4% at 96 h (Fig. 1A , P<0.01, n=3–11). Impaired wound healing and amplified PMN recruitment in response to topical LPS treatment correlated with a ~4-fold increase in the chemokines KC (P<0.03, n=5) and MIP2 (P<0.003, n=5), and a ~6-fold increase in the cytokine IL-6 (P<0.02, n=5) content of mouse corneas 48 h after injury (Fig. 1C ), consistent with an exacerbated inflammatory response.


Figure 1
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  		Figure 1. Exacerbated inflammation impairs epithelial wound healing. Mouse eyes were treated topically with LPS (10 µg, t.i.d., hashed bars) or saline alone (solid bars) for 24–96 h after epithelial injury. A) Corneas were collected and neutrophil content determined by measuring myeloperoxidase activity (n=3–8, *P<0.05 saline vs. LPS). B) Wound healing was assessed by fluorescein staining of the de-epithelialized area and digital image analyses (n=3–11, P<0.05 *24, 48 vs. 96 h, # saline vs. LPS). C) Corneas were dissected from saline or LPS-treated eyes and homogenized 48 h post-injury. KC (mouse IL-8 homologue), MIP-2, and IL-6 content was quantified using a specific custom proteome microarray (n=5, *P<0.05).

12/15-LOX is highly expressed in corneal epithelial cells (7 , 12 , 13 , 15) and initiates the formation of LXA4 whose G-protein coupled receptor is expressed in both murine leukocyte and corneal epithelial cells (50 , 51) . Topical treatment of injured mouse corneas with LXA4 significantly attenuated formation of the chemokine KC by 35–67% at doses of 1–1000 ng (t.i.d) 48 h after injury (Fig. 2 A, P<0.04, n=6–10). By contrast, 15-HETE, a metabolic precursor for LXA4, did not inhibit KC formation at a topical dose of 1–1000 ng (t.i.d., n=4–6). We next assessed whether LXA4 could counter-regulate LPS-induced exacerbated inflammation and restore normal wound healing. LXA4 treatment (1 µg, t.i.d.) significantly reduced LPS-induced KC formation (n=5, P<0.03) and PMN infiltration (n=11–17, P<0.001) by 64% and 54%, respectively (Fig. 2B and C ). More importantly, topical treatment with LXA4 counter-regulated the negative action of LPS on wound healing by increasing the rate of re-epithelialization by 83 ± 18% (Fig. 2C , n=11, P<0.001), 48 h after injury.


Figure 2
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  		Figure 2. Topical LXA4 protects against exacerbated inflammation and restores wound healing. A) Eyes were treated topically (1–1000 ng, t.i.d.) with LXA4 (n=6–10), 15-HETE (n=4–6), or saline alone for 48 h after epithelial injury. Corneas were collected and homogenized, and KC content was quantified using a custom proteome microarray. *Different from saline (P<0.04). B) Injured corneas were treated (t.i.d.) with LPS alone or a solution containing both LPS (10 µg) and LXA4 (1 µg). Forty-eight hours after injury, corneas were collected and KC content was quantified (n=3–5). *Different from LPS alone (P<0.05). C) PMN content in corneas treated for 48 h with LPS or LPS and LXA4 was determined by measuring myeloperoxidase activity as a quantitative PMN marker (n=11–17). Corneal wound healing at 48 h post-injury was assessed by fluorescein staining and digital image analyses (n=4–5). *Different from LPS alone (P<0.001).

To establish a direct role for the 12/15-LOX pathway in counter-regulating exacerbated inflammation and impaired wound healing, we utilized the well characterized 12/15-LOX knockout mice (Alox15–/–) (15 , 17) , which have impaired formation of LXA4 in the cornea (7) . In these Alox15–/– mice wound healing in LPS challenged corneas was characterized by a 43% increase in corneal PMN content (n=10, P<0.04) and a 29% decrease in the rate of wound closure 48 h after injury (n=14, P<0.05) when directly compared to LPS-treated matched congenic controls (Fig. 3 A). Endotoxin treatment dramatically reduced the rate of wound healing in Alox15–/– mice by 68% when directly compared to the saline-treated Alox15–/– controls (18% vs. 47%, P<0.002, n=3). Topical treatment with the 12/15-LOX pathway metabolite LXA4 partially restored the rate of normal healing in these LPS challenged wounds to 78% of the no endotoxin-treated Alox15–/– controls (P<0.024, n=3). Taken together, data from the 12/15-LOX knockout mice and the rescue experiments with LXA4 provide strong evidence that endogenous 12/15-LOX-derived lipid autacoids play a critical role in counter-regulating exacerbated inflammation during epithelial wound healing.


Figure 3
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  		Figure 3. Deletion of LXA4 biosynthetic pathway is associated with exacerbated inflammation and impaired wound healing. A) 12/15-LOX–/– mice and their matched congenic controls were subjected to corneal epithelial injury. Eyes were treated topically (t.i.d.) with LPS (10 µg) for 48 h after injury. PMN content in corneas was determined by measuring myeloperoxidase activity (left panel, n=10, *P<0.05). Epithelial wound healing was assessed by fluorescein staining and digital image analyses (right panel, n=14, *P<0.05). B) 12/15-LOX–/– mice were treated topically (t.i.d.) with saline, LXA4 (1 µg), and/or LPS for 48 h after epithelial injury. Wound healing was assessed by digital image analyses (n=3, *P<0.05, different from saline alone and LXA4 plus LPS). Inset representative images (Saline alone, LPS alone, or LXA4 plus LPS group) of fluorescein-stained corneas 48 h post-epithelial injury.

We have recently demonstrated that the cytoprotective heme-oxygenase system plays a critical role in the execution and resolution of an acute inflammatory/reparative response in the cornea (11) . To determine if deletion of a key antiinflammatory lipid mediator pathway impacts the induction or expression of the corneal heme-oxygenase system, we assessed protein expression of HO-1 in Alox15–/– mice in response to injury and endotoxin challenge. Epithelial injury induced consistent and robust de novo expression of HO-1 48 h post epithelial injury (Fig. 4 A). Furthermore, treatment of epithelial wounds with LPS significantly amplified HO-1 protein expression by 3.0 ± 0.5-fold compared with saline treated corneal wounds in wild-type mice (Alox15+/+). In sharp contrast, HO-1 induction in Alox15–/– mice was significantly impaired by ~3-fold (Fig. 4A , P<0.02, n=3) in both the basal inflammatory response to epithelial injury and the exacerbated inflammatory response evoked by LPS challenge. We next assessed if impaired HO-1 expression in Alox15–/– mice can be restored by adding back an endogenous bioactive 12/15-LOX metabolite, namely LXA4. Western blot analyses shown in Fig. 4B demonstrate topical treatment with LXA4, indeed, partially restored HO-1 protein expression in Alox15–/– mice. These data provide the first evidence for a potential functional link between two prominent antiinflammatory circuits, namely 12/15-LOX and HO-1.


Figure 4
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  		Figure 4. Impaired induction of the cytoprotective HO-1 circuit in 12/15-LOX–/– mice. A) 12/15-LOX–/– and congenic control mice were subjected to epithelial injury and treated with saline or LPS (10 µg) for 48 h. Expression of HO-1 in corneas was analyzed by Western immuno-blot analyses. Inset shows representative analyses from 2 mice per data point. Bar graph shows densitometry analyses after HO-1 expression was normalized to ß-actin. Data are expressed as fold change from injured WT (n=8–10, ANOVA, *P<0.05, difference among groups). B) LXA4 treatment partially restores HO-1 expression in 12/15-LOX–/– mice. Eyes were treated topically with saline or LXA4 (1 µg, t.i.d.) for 48 h post-epithelial injury. Inset shows representative Western immuno-blot analyses from 2 mice per treatment. Bar graph shows densitometry analyses (n=4, *P<0.05).

Epithelial cells play a key role in inflammation and wound healing and, in response to corneal injury, are a predominant cell type for the expression of HO-1. Hence, we investigated, in vitro, if products of the 15-LOX pathway can regulate HO-1 expression in human corneal epithelial cells. Human corneal epithelial cells were challenged with selected arachidonic acid and docosahexaenoic acid derived 15-LOX metabolites, since these lipid autacoids are formed in the injured cornea and promote epithelial wound healing (7) . Quantitative real-time PCR analyses demonstrated that LXA4, 17S-HDHA, and NPD1 (10R,17S-dihydroxy DHA) significantly increased basal HO-1 mRNA expression ~1.4- and 2.0-fold at a concentration of 10 and 100 nM, respectively (Fig. 5 A, P<0.009, n=5). By contrast, 15S-HETE, a LXA4 metabolic intermediate did not increase basal HO-1 mRNA expression in human corneal epithelial cells and at 100 nM significantly decreased basal HO-1 expression. RT-PCR analyses confirmed that the human corneal epithelial cell line expresses both 15-LOX genes (ALOX15 and ALOX15B) and the LXA4 receptor, ALX (Fig. 5B ).


Figure 5
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  		Figure 5. Antiinflammatory lipid mediators of the 15-LOX pathway induce expression of epithelial HO-1. A) Real-time PCR analyses for HO-1 mRNA expression in human corneal epithelial cells. Cells were subjected to 12 h of starvation followed by 8 h of treatment with vehicle or indicated lipid mediators. Data (n=4) is expressed as the log-fold change in HO-1 mRNA expression (Pfaffl method) compared to the calibrator (cells without treatment) and normalization to the reference gene GAPDH. B) Representative (n=3) RT-PCR demonstrating mRNA expression of 15-LOX (ALOX15 and ALOX15B) and LXA4 receptor (ALX).

Data in Figs. 4 and 5 suggest a potential functional link between the 15-LOX system and HO-1 systems. These findings are of particular interest since in vivo deletion of HO leads to chronic inflammation and impaired wound healing (11 , 31 , 35) . However, the impact of disrupting the heme-oxygenase system on antiinflammatory lipid mediator circuits has not been investigated. Hence, corneas of HO-2 knockout mice and their matched wild-type controls were subjected to epithelial injury and endogenous LXA4 formation was measured. Consistent with our previous report (11) , inflammation was dramatically amplified in HO-2 knockout mice as evidenced by massive and sustained infiltration of PMN 2 and 4 d post-epithelial injury and pronounced neovascularization at day 7, which was completely absent in wild-type mice (Fig. 6 A). This phenotype of chronic inflammation, impaired wound healing and neovascularization, which we recently fully characterized (11) , correlated with a consistent ~50% decrease in endogenous LXA4 formation in the injured cornea of HO-2–/– mice (Fig. 6B ) at 24, 48, 72, and 96 h after epithelial injury when directly compared with matched wild-type mice. Taken together, in vivo loss of function experiments with 12/15-LOX–/– and HO-2–/– mice provide strong evidence for the interdependence of the HO and 12/15-LOX system in the acute inflammatory/reparative response of the cornea.


Figure 6
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  		Figure 6. Heme-oxygenase deficient mice exhibit a phenotype of chronic inflammation and neovascularization associated with impaired LXA4 formation. HO-2 knockout mice (HO-2 and their matched wild-type controls were subjected to corneal epithelial injury. A) At days 2 and 4 post-injury, corneas were collected, fixed, and sectioned, and 10 µm sections were stained with hematoxylin/eosin. Representative (400x, n=4) histology of corneas from WT and HO-2–/– mice. At day 7 neovascularization was assessed by slit-lamp microscopy and digital image analyses. Shown is a representative image (n=12) of WT and HO-2–/– eye. B) Corneas were collected from WT mice (1, 2, 3, 4, and 5 d) and HO-2–/– mice (1, 2, 3, and 4 d) after epithelial injury, homogenized in ice-cold MeOH and extracted, and LXA4 content was analyzed by a specific ELISA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Physical injury is often associated with exposure of the denuded tissue to bacteria and/or bacterial products, which are potent activators of the innate host defense and, subsequently, amplify basal inflammation associated with the reparative response. Clearly, inflammation and recruitment of leukocytes are critical to host defense and essential for survival. However, the role of inflammation and leukocyte cell types in the tightly orchestrated cascade of wound healing and resolution is far from clear (2) . A wealth of research underscores how difficult it is to dissociate these intimately linked innate responses and provides evidence that the state of leukocyte activation, degree and resolution of the inflammatory response, likely dictate whether inflammation is beneficial or detrimental to the wound healing process.

In this regard, the cornea is of special interest as it is an avascular and immune-privileged environment where inflammation is actively suppressed and injuries induced by surgical procedures or epithelial abrasion heal rapidly with almost no scarring. Moreover, recent reports demonstrate that inflammation and PMN recruitment in the cornea is beneficial for wound healing (7 8 9 10) . However, even in the cornea, severe injury induces exacerbated inflammation, which is associated with impaired wound healing (52) . Mechanisms that account for the striking properties of the cornea, which appear to be absent in most other adult tissues, remain to be defined. 12/15-LOX may constitute one such mechanism; these important immune-modulatory enzymes are highly expressed in mouse and human corneas (7 , 12 , 13) and generate potent antiinflammatory lipid autacoids in healthy and injured corneas (7) . This hypothesis is supported by results obtained from two independent experimental approaches: 1) Exacerbated inflammation and impaired wound healing (Fig. 1) are attenuated by pharmacological amplification of the 12/15-LOX pathway, namely topical treatment with LXA4, which inhibits formation of the chemokine KC and recruitment of PMN and restores normal wound healing (Fig. 2) ; and 2) Genetic deletion of a key LXA4 biosynthetic pathway (7) , 12/15-LOX, further amplifies exacerbated inflammation and impairs wound healing and, consistent with the hypothesis, adding back LXA4 attenuates the exacerbated inflammation and partially restores wound healing (Fig. 3) .

12/15-LOX (ALOX15 in Humans and Alox15 in mice) has pleiotropic effects in inflammatory diseases (15 , 17 , 20) ; however, numerous reports provide strong evidence that the primary 12/15-LOX metabolites from arachidonic acid (15-HETE and LXA4) and docosahexaenoic acid (17-HDHA and NPD1) are antiinflammatory (19 , 22 , 24) and neuroprotective (16 , 29) . Moreover, we recently identified a novel bioaction for 12/15-LOX and its endogenous metabolites, LXA4, 17-HDHA, and NPD1, in promoting epithelial wound healing and limiting the sequelae of corneal injury, which is distinct from their well established antiinflammatory properties (7) . Indeed, quite contrary to their established bioaction, topical treatment with endogenous antiinflammatory lipid autacoids significantly increased PMN content in the cornea, and genetic deletion of 12/15-LOX, which resulted in 50% decrease of endogenous LXA4, reduced PMN content in the injured cornea. Hence, our findings (7) and independent reports from other investigators (8 9 10) support the notion that controlled inflammation has a beneficial role in epithelial wound healing in the cornea. The observation that 12/15-LOX metabolites inhibit PMN recruitment when inflammation is exacerbated (Figs. 2 and 3) , but do not impair PMN migration to a site of injury where acute inflammation and the reparative response are balanced (7) , underscores the primary role of this pathway in counter-regulating excessive PMN activation rather than inhibiting the beneficial activation of these primary effector cells. Hence, the atypical constitutive formation of antiinflammatory mediators in the cornea may provide a unique setting for the wound healing process to constrain PMN activation, which is likely absent in most other injured tissues unless the basal tone of antiinflammatory mediators is amplified.

Therefore, it is of particular interest that an extensive epithelial abrasion in the cornea, which is characterized by self-resolving inflammation and complete wound healing in 5–7 d, shifts to a chronic inflammatory scenario marked by massive neovascularization and failure to heal if constitutive expression of the cytoprotective heme-oxygenase system is disrupted (Fig. 6) . These HO-2 knockout mice exhibit markedly impaired induction of the stress gene HO-1 in response to epithelial injury, which leads to a phenotype of chronic inflammation (11) , emphasizing the established antiinflammatory role of the heme-oxygenase system (31) . It is striking that both 12/15-LOX and heme-oxygenase are highly expressed in the injured cornea (11 , 14 , 50) and that disruption of either one of the genes exacerbates inflammation and delays wound healing (Figs. 3 and 6) , which provides in vivo evidence that both resident antiinflammatory systems are crucial to counter-regulating proinflammatory pathways.

Here, we present the first evidence that these two protective systems function in concert to control inflammation. This conclusion is supported by experiments that demonstrate that 12/15-LOX metabolites LXA4, 17-HDHA, and NPD1 amplify HO-1 expression in epithelial cells (Fig. 5) . Note that 15S-HETE, a primary product of 12/15-LOX and metabolic intermediate for LXA4, did not amplify HO-1 expression in epithelial cells, which is consistent with the observation that 15-HETE, unlike LXA4, does not inhibit KC formation in the cornea. However, 15-HETE did increase the rate of epithelial wound healing at a topical dose of 1–1000 ng (data not shown) and is an intracellular second messenger in corneal epithelial cells for the epidermal cell growth factor (53) . These findings provide compelling evidence that the mechanism of action for 15S-HETE to promote wound healing is distinct from that of LXA4, 17S-HDHA, and NPD1. In addition, unlike 15S-HETE, the docosahexaenoic acid-derived metabolites 17S-HDHA and NPD1 are both potent antiinflammatory lipid mediators and promote wound healing (27 , 41 , 50) , which is consistent with their ability to induce HO-1 (Fig. 5) . The physiological relevance of these in vitro findings is strongly supported by experiments with 12/15-LOX knockout mice as these mice generate 50% less LXA4 in response to corneal injury compared to their congenic controls (7) , which correlates with impaired induction of HO-1 in both acute and exacerbated inflammation (Fig. 4) . Consistent with the hypothesis that LXA4 amplifies endogenous HO-1 expression, topical treatment with LXA4 restored HO-1 expression in injured corneas of the 12/15-LOX knockout mice (Fig. 4) .

HO-2 knockout mice exhibit a dramatic phenotype of an aberrant inflammatory/reparative response whose hallmarks are unabated infiltration of leukocytes and failure to resolve an acute inflammatory event, both of which are key targets for the bioaction of endogenous LXA4. Thus, it is striking that endogenous LXA4 formation was consistently reduced by 50% in injured corneas of HO-2 knockout mice (Fig. 6) , while levels of proinflammatory eicosanoids remained highly elevated (11) . Heme–oxygenase generates two prominent protective mediators: 1) the antioxidant bilirubin; and 2) the potent gas carbon monoxide, which activates guanylate cyclase and regulates MAP kinase pathways causing down-regulation of the proinflammatory cytokines IL-6, TNF-{alpha}, IL-1ß, and MIP-1{alpha} (31 , 35) . It is important to point out that numerous reports have demonstrated in vitro and in vivo that LXA4 mimetics down-regulate the same profile of proinflammatory cytokines (54 55 56) , which are the targets for the protective action of carbon monoxide. Hence, the molecular mechanisms for the interdependence of the HO and 12/15-LOX are of considerable interest and the subject of ongoing research efforts.

In summary, the current findings demonstrate a crucial role for resident antiinflammatory systems in controlling inflammation in order to maintain wound healing and suggest an interdependence of the HO and 12/15-LOX circuits. In view of these results and the striking complementary protective actions of these pathways in regulating acute inflammation, it is tempting to speculate that interaction of the 12/15-LOX and HO-1 systems provides a positive feedback loop that amplifies antiinflammatory signals. Amplification of antiinflammatory circuits is likely critical to counterbalance proinflammatory cascades, whose tightly regulated activation is essential for execution of acute inflammatory/reparative responses without damaging the delicate visual axis.


   ACKNOWLEDGMENTS
 
We thank Melody Steinberg for editing the manuscript. This work was supported in part by National Institutes of Health grants EY016136 (KG) and EY06513 (MLS).

Received for publication December 14, 2006. Accepted for publication February 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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