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Int J Exp Pathol. Apr 2009; 90(2): 166–173.
PMCID: PMC2676704

The lectin KM+ induces corneal epithelial wound healing in rabbits

Abstract

Neutrophil influx is essential for corneal regeneration (Gan et al. 1999). KM+, a lectin from Artocarpus integrifolia, induces neutrophil migration (Santos-de-Oliveira et al. 1994). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6.0-mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 μg/ml) every 2 h. The control eyes received buffer. The epithelial wounded areas of the lectin-treated and untreated eyes were stained with fluorescein, photographed and measured. The animals were killed 12 h (group 1, n = 5), 24 h (group 2, n = 10) and 48 h (group 3, n = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p63, vascular endothelial growth factor, c-Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls. Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.

Keywords: Artocarpus integrifolia, corneal wound healing, KM+ lectin, neutrophils

Corneal epithelial defects represent a relatively frequent clinical problem in ophthalmology and may be related to different conditions such as refractive surgery, contact lens wear, bullous keratopathy, corneal exposure, dry eye, chronic dacryocystitis, immunosuppressive agents, corneal infections, chemical burn or trauma (Dawson et al. 2005). In addition, the loss of the corneal epithelium integrity predisposes to bacterial or fungus keratitis, a serious eye condition that may lead to vision loss if untreated (Mitchell & Wilhelmus 2005).

The corneal epithelium acts as a barrier between the external and intraocular environments and its integrity and transparency are essential for corneal physiology (Gipson & Sugrue 1994; Zieske & Gipson 1994). For these reasons, the search for effective treatment for corneal wounds should aim at prompt repair of disrupted surface epithelium, restoration of corneal integrity and preservation of visual function (Kruse 1994; Schultz et al. 1994). Although several substances have been reported to induce corneal epithelial regeneration (Macsai 2000), corneal wound healing remains a clinical problem in ophthalmology.

A large number of growth factors and their associated receptors, including epidermal growth factor (EGF), keratinocyte growth factor, hepatocyte growth factor (HGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) have been shown to have an effect on corneal wound repair (Klenkler & Sheardown 2004). Therefore, substances that induce growth factors, directly or indirectly, are able to speed up corneal reepithelization.

A close relationship has also been observed between the presence of neutrophils and production of growth factors. Indeed, neutrophils are a source of growth factors and cytokines, all of which are necessary for initiation and propagation of new tissue formation in wounds (Baird et al. 1985). In addition, neutrophils have been shown to produce several proangiogenic cytokines, including vascular endothelial growth factor (VEGF), tumour necrosis factor α, interleukin (IL)-1, IL-6 and IL-8 (Dubravec et al. 1990; Liles & Van Voorhis 1995; Ericson et al. 1998; McCourt et al. 1999). Some of them were described as overexpressed during intense epithelial proliferation process. On the other hand, blocking of neutrophil influx has been associated with decreased epithelial healing rate in injured cornea (Gan et al. 1999). Furthermore, the epithelium proliferative index, seen as proliferation cell nuclear antigen (PCNA) expression, is also reduced in the absence of neutrophils (Gan et al. 1999).

KM+, a structurally well defined d-mannose-binding lectin from Artocarpus integrifolia seeds, also known as artocarpin, is able to induce neutrophil migration both in vivo and in vitro (Santos-de-Oliveira et al. 1994). As a tetrameric molecule, KM+ has four carbohydrate recognition domains, one per each protein chain (Rosa et al. 1999). It allows the molecule to establish concomitant interactions with the neutrophil surface glycans, such as those associated with CXCR2 (Pereira-da-Silva et al. 2006) and with glycosylated components of the extracellular matrix (ECM), such as laminin (Ganiko et al. 2005). The reversible bridge formed by KM+ between neutrophils and the matrix of the perivascular tissue is probably responsible for the neutrophil haptotaxis induced in vivo by the lectin (Ganiko et al. 1998). In addition, the KM+ binding to neutrophil receptors promotes cell activation, which results in increased Toll-like receptors (TLRs) expression and releasing of cytokines, such as IL-8 (Roque-Barreira 2008). Considering the relevant role of neutrophils in the corneal regeneration and the KM+ property of inducing neutrophil migration and activation, we have investigated the effect of KM+ on corneal epithelial wound healing.

Materials and methods

Animals

Male albino rabbits of the New Zealand strain weighing 2.0–2.5 kg and with no signs of ocular inflammation or gross abnormality were used. The animals were housed in individual cage, under standard conditions of temperature and 12:12 h light/dark cycle, with food provided ad libitum. Animal management procedures conformed to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research and the International Guiding Principles for Biomedical Research Involving Animals. The rationale, design and methods of this study were approved by our institutional animal ethics committee (no. 106/2007). Efforts were made to minimize animal suffering and to reduce the number of animals used. To exclude any disease that could interfere with the re-epithelization process, eyes of all animals were previously examined with a slit lamp. Animals were randomly divided into two groups, one consisting of five rabbits and other of 10 rabbits. They were kept in restraining boxes only during eye manipulations.

Drugs, surgery and treatment

The lectin KM+ was purified by affinity chromatography as previously described by Santos-de-Oliveira et al. (1994). The lectin homogeneity was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and protein concentration was measured by the method of Lowry et al. (1951). KM+ was diluted at a concentration of 2.5 μg/ml under sterile conditions in a mucoadhesive, isotonic (in respect to the rabbit tear tonicity), and buffered (pH 7.4) topical formulation. The vehicle alone was used as a control preparation.

The rabbits were anesthetized with an intramuscular injection of ketamine (Ketamine; Parke-Davis Inc., Morris Plains, NJ, USA), at a dose of 40 mg/kg. Additional topical anaesthesia was carried out with one drop on the conjunctiva of each eye of 0.5% proxymetacaine hydrochloride commercial preparation. Analgesia was performed with oral sodic dipirone (1 drop/kg) every 6 h.

The debridement of the corneal epithelium was performed as follows: The centre of the cornea was marked with a 6-mm-diameter trephine and afterwards the epithelium inside this circle was scraped with a surgical blade no. 15, until it reached the basement membrane which was removed with the epithelium (Johansen et al. 1998). A uniform, round lesion was produced in each eye, which without treatment, remains opened until 24 h. The eyes were then carefully rinsed with saline (50 ml). This surgical procedure was performed on both eyes of the 20 rabbits. KM+ solution, 2.5 μg/ml, was dropped on the right eyes at intervals of 2 h, during 12 h, whereas the respective left eyes received the vehicle only (buffer), one drop every 2 h, until the time they were killed. The five rabbits of group I were killed at 12 h, the 10 of group II at 24 h and the five of group III at 48 h after debridement.

Epithelial corneal wound healing assessment

The progression of epithelial corneal wound healing was evaluated by staining with fluorescein followed by immediate photography (Bonfiglio et al. 2006) with a tripod stabilized 35-mm camera (Nikon, Tokyo, Japan) equipped with a 1:1 macrofocus lens, positioned directly in front of the animal's eye, at 12, 24 and 48 h after epithelial abrasion. The sizes of the epithelial defects were evaluated independently in each eye and were determined in mm2 using a standard software (image j). Measurements were compared between KM+ treated and untreated eyes.

Histological assessment

Animals were killed using a CO2 chamber. Corneas were excised, fixed in 10% phosphate-buffered formalin for 24 h at room temperature and then embedded in paraffin. The sizes of the epithelial defects and neutrophils influx were assessed in 5 μm sections, which were stained with haematoxylin and eosin. Samples were blindly evaluated by two of the authors (FC and LZR). All discordant results were resolved using a multi-head microscopic analysis system. The corneas of animals which presented remaining epithelial defects at the time of killing (groups 1 and 2) were also submitted to immunohistochemical analysis.

For immunohistochemical analysis, 4-μm-thick sections mounted on poly-l-lysine-coated slides were deparaffinized, rehydrated, immersed in 10 mmol/l citrate buffer, pH 6.0, and submitted to heat-induced epitope retrieval using a vapour lock for 45 min. The slides were briefly rinsed with phosphate-buffered saline (PBS) and immersed in 3% hydrogen peroxide for 20 min to block endogenous peroxidase. Non-specific protein binding was blocked with normal serum (Novostain Universal Super ABC Kit; Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) for 30 min. The sections were then incubated with monoclonal primary antibodies specific for proliferating cell nuclear antigen (PCNA, clone PC10; DAKO A/S, Denmark, dilution 1:100), p63 (clone 4A4; Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:400), VEGF (clone A20; Santa Cruz Biotechnology, dilution 1:400), transmembrane receptor tyrosine kinase c-Met (clone 8F11; Novocastra Laboratories Ltd, dilution 1:100) and laminin (clone LAM89; Novocastra Laboratories Ltd, dilution 1:100), for 2 h at room temperature (~25 °C) in a humid chamber. Following washes in PBS, biotinylated pan-specific universal secondary antibody (Novostain Universal Super ABC Kit; Novocastra Laboratories Ltd) was applied for 30 min. Next, the slides were incubated with the avidin–biotin–peroxidase complex (Novostain Universal Super ABC Kit; Novocastra Laboratories Ltd) for 30 min and developed with 3,3-diaminobenzidine tetrahydrochloride (DAB) (Novocastra) in PBS for 5 min. The slides were counterstained by Harris haematoxylin, dehydrated and mounted with Permount (Biomeda, Foster City, CA, USA). As negative controls, all specimens were incubated with an isotope-matched control antibody under identical conditions.

The preparations for each marker were evaluated randomly, at least 10 representative high-power fields (×40), by two of the authors (FC and LZR). All discordant results were resolved using a multi-head microscopic analysis system. The immunolabelling percentage was evaluated by a ratio of unequivocal labelling located at nuclei (PCNA and p63), membrane (laminin), nuclei/cytoplasm (c-Met) or cytoplasm (VEGF) at each 100 counted epithelial cells. However, only non-ulcerate areas of the cornea epithelium (lesion periphery) were compared by immunohistochemistry.

Statistical evaluation

All data were analysed using the graphpad prism software 4.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical comparisons were carried out using the Wilcoxon test and two-sided Fisher's exact test, as appropriate. A P value < 0.05 was required for significance.

Results

The epithelial resurfacing was incomplete in all eyes of group 1 (12 h after debridement), and the gap between the sliding fronts was practically of the same dimensions in control and KM+-treated eyes (P > 0.05) (Table 1). In this same group, there was an evident difference between control eyes and KM+-treated eyes concerning the neutrophil influx. The number of neutrophils/unit area was significantly higher in KM+-treated corneas (254.2 neutrophils/mm2 ± 0.2) than in the control ones (176 neutrophils/mm2 ± 0.5) (P <0.05) (Table 1). These cells were concentrated in the stroma at the region of the sliding epithelial front which was made up by a single layer of cells (Figure 1a,b).

Table 1
Effects of KM+ treatment on epithelial defect size, ulcer presence and polymorphonuclear (PMN) influx, after 12, 24 and 48 h of corneal abrasion in rabbits
Figure 1
Histological appearance of corneas that underwent abrasion and were treated with topical KM+ (a) or buffer (b) at 12 h (magnification: ×20). Histological appearance of corneas that underwent abrasion and were treated with topical KM+ (c) or buffer ...

Although no cornea was healed 12 h after debridement (group 1), 24 h after (group 2) the gap was closed in seven of the 10 treated eyes and only in three of the 10 control ones. The epithelial gaps were significantly smaller in KM+-treated corneas than in the untreated ones (1.7 ± 0.1 vs. 4.5± 0.2 mm2, respectively) (P <0.05) (Table 1). Only rare neutrophils were observed in the corneal stroma of group 2, in both treated and control eyes (Figure 1c,d). No epithelial gaps, neutrophils influx or stroma enlargement were observed both in KM+-treated corneas and the control ones (Figure 1e,f).

No significant reaction with the antibodies specific for PCNA, p63, VEGF, c-Met and laminin was detected in any of the eyes of group 1, whereas 24 h after debridement (group 2) the percentage of the corneal epithelial cells labelled with the same antibodies was higher in the lectin-treated eyes (47.4 ± 0.4; 46 ± 0.5; 29.5 ± 0.7; 74.5 ± 0.6; 42.5 ± 0.5, respectively) than in the untreated ones (27.6 ± 0.2; 5.7 ± 0.4; 2.4 ± 0.5; 7.3 ± 0.4; 4.8 ± 0.5, respectively) (P < 0.05) (Table 2).

Table 2
Effects of KM+ treatment on proliferation cell nuclear antigen (PCNA), p63, vascular endothelial growth factor (VEGF), c-Met and laminin labelling, after 12 and 24 h of corneal abrasion in rabbits

The PCNA expression was observed in the nuclei of epithelial cells (Figure 2a,b). At least two cell layers were visualized in most of the resurfaced stroma. The p63 protein, similarly to the PCNA antigen, was revealed in the nucleus (Figure 2c,d), VEGF in the cytoplasm (Figure 2e,f) and c-Met (Figure 2g,h) in the nucleus/cytoplasm of epithelial cells. Laminin (Figure 2i,h) was detected in the epithelial cellular membranes at 24 h in the KM+-treated eyes. Laminin expression was also observed in the basement membrane of lesion periphery and regenerated epithelium, but this finding was not considered for comparative analysis. VEGF was restricted to the superficial layers whereas c-Met and laminin were widespread through all layers of the regenerated corneal epithelium.

Figure 2
Immunohistochemical labelling of corneas that underwent abrasion and were treated with topical KM+ or buffer at 24 h for proliferation cell nuclear antigen (a and b); P63 (c and d); vascular endothelial growth factor (e and f); laminin (g and h) and c-Met ...

Discussion

The corneal epithelium is the front barrier against the outside lethal agents to the eye. Therefore, nature has endowed this epithelium with an extremely efficient mechanism for a quick regeneration in case of damage to the eye surface. In fact, a combination of cell sliding and proliferation enables the epithelium to cover a wounded area, in a very short time, after epithelial loss (Kuwabara et al. 1976; Zieske et al. 2004). Inflammation is also a reaction to corneal epithelial damage (Wilson et al. 2001) and according to widely accepted basic tenets, the immediate response of the body, as represented by eye acute inflammatory process, involves changes in calibre and structure of small blood vessels as well as migration of neutrophils to the extravascular environment (Xue et al. 2007). This concept is supported by the demonstration that blockage of neutrophil migration to the site of injury results in a delay of the corneal epithelium resurfacing (Gan et al. 1999).

Among the biological properties of the KM+ lectin, the induction of neutrophil migration and activation was verified by investigation on its mechanism of action (Santos-de-Oliveira et al. 1994; Ganiko et al. 1998, Ganiko et al. 2005; Pereira-da-Silva et al. 2006) and the cellular functional repercussions of KM+ stimulus (Roque-Barreira 2008). In this study, we observed a neutrophil influx into the corneal stroma 12 h after abrasion, a phenomenon that was enhanced in the KM+-treated corneas. Moreover, our finding may indicate that the neutrophil accumulation in the wounded area is more relevant at an early phase of corneal repair, as previously demonstrated by other authors (O’Brien et al. 1998; Wilson et al. 2001).

Neutrophil influx might be a primordial event in corneal wound healing as this was the most striking morphological finding on light microscopy 12 h after abrasion, along with the epithelial defect. In sequence, the inflammatory infiltrate vanish as epithelial proliferation begins, with the restoration of corneal epithelium in most treated corneas 24 h after abrasion (Zhijie et al. 2006). A modulation mechanism of neutrophil influx seems to occur during corneal wound healing both in KM+-treated and in control eyes, as neutrophils in the corneal stroma were not observed at 24 h after abrasion. Thus, KM+ did not stimulate a continuous corneal neutrophil accumulation, which could result in the worsening of corneal injury. The time interval between the neutrophil infiltration and the epithelial recovery may correspond to the period of epithelial proliferation due to cytokines and growth factors released by neutrophils, before they underwent to apoptosis (Shaw et al. 2003). In addition, the phagocytosis of apoptotic neutrophils leads to secretion of cytokines related to wound healing (Golpon et al. 2004).

The peripheral neutrophil, which has been considered as a terminal cell until some years ago, is now recognized as capable of transcripting a certain number of genes whose production lie at the core of inflammatory response (Scapini et al. 2000; Cloutier & McDonald 2003), such as genes of cytokines, chemokines, growth factors and surface receptors (Shaw et al. 2003; Sabroe et al. 2005; McGettrick & O’Neill 2007; Nakanaga et al. 2007). Under KM+ stimulation, neutrophil enhances such a capacity as demonstrated by the detection of augmented levels of IL-8 production and mRNA encoding TLR2 (Roque-Barreira 2008). The high expression of several other substances which, once present at the site of corneal wounding, stimulate or accelerate the healing can also be included in the response to KM+-induced cell activation, a hypothesis coherent with the observation that a larger number of neutrophils at 12 h in the KM+-treated eyes was the distinct morphological feature detected in the corneal microscopy, compared to control eyes. Therefore, the acceleration of the resurfacing, as observed in group 2 KM+-treated eyes, can result from the stimulation caused by the neutrophil products (Gan et al. 1999).

There is recent evidence that neutrophils produce several proangiogenic factors, including VEGF (Dubravec et al. 1990; Liles & Van Voorhis 1995; Ericson et al. 1998) and others (FGF, TGF-β and PDGF). VEGF refers to a family of angiogenic and vascular permeability-enhancing peptides derived from alternatively spliced mRNAs that can act as an important factor in the cascade leading to the onset of corneal neovascularization (Wolfgang et al. 2000). It was shown that VEGF expression in the human corneal epithelium is enhanced following ocular surface trauma (Van Setten 1997). In addition, VEGF is active in the wound-healing phase and is expressed in the proliferating epithelium, keratocytes and endothelium (Gan et al. 2004). We found, 24 h after abrasion, a higher VEGF expression in KM+-treated cornea. This observation reinforces the idea that KM+ may act as a stimulator of corneal re-epithelization, a fact that may be related to the augment of VEGF expression in corneal epithelial cells during the corneal restoration.

The expression of the PCNA protein, which is related to the S-phase of the cell cycle, was highest in the corneas treated with KM+ for 12 h and examined 24 h after the onset of the treatment. Under physiological conditions, there is a very low percentage of DNA-synthesizing corneal epithelial cells at short intervals such as 12 or 24 h (Szerenyi et al. 1994; Haddad 2000). This is also true when the epithelium is mobilized to cover the scraped area because the main early event is sliding and not proliferation (Kuwabara et al. 1976; Zieske et al. 2004). Proliferation is notable only after 12–24 h postdamage (Cotsarelis et al. 1989). Increased expression was also observed for p63, which is essential for proliferation and differentiation in stratified epithelia (Blanpain & Fuchs 2007). The neutrophil products released in the site of epithelial damage can be responsible for the higher PCNA and p63 expressions and the cell proliferation observed in the treated and resurfaced eyes of group 2. Nevertheless, a direct effect of KM+ in stimulating cell corneal epithelium proliferation cannot be excluded.

The neutrophil migration induced by KM+ involves the lectin interaction with ECM components, above all with laminin, which promotes the formation of a substrate-bound gradient of KM+ necessary for the haptotatic movement of neutrophils that transmigrate through the vascular wall (Ganiko et al. 2005). In this study, laminin immunostaining was not detected intracellularly in the corneas of KM+-treated or non-treated eyes, at 12 h after abrasion. However, at 24 h of injury, laminin was detected in the regenerating epithelium and interface epithelium-stroma, in the KM+-treated eyes, a fact that is consistent with the important role attributed to the ECM components on the effective action of KM+ regarding the neutrophil migration and, putatively, the cornea wound repair. During the corneal epithelium regeneration the re-establishment of the cell junctions precedes the formation of new basement membrane (Hutcheon et al. 2007) which takes about a week to exhibit a continuous profile. The intracellular location of laminin and its discontinuity and unevenness in the epithelium-stroma interface fit the reconstruction of the basement membrane. The demonstration of the intracellular location of laminin is noteworthy as it is rarely observed in adult cells (Laurie et al. 1980; Murray & Leblond 1988). We cannot keep out the possibility that the immunohistochemical visualization of the intracellular laminin may be a consequence of the protocol used in our investigation, which is assumed to expose the antigen epitopes more than the fixation by freezing and sectioning in a cryostat (Hutcheon et al. 2007).

Our results also demonstrated the overexpression of the HGF receptor (c-Met) exclusively in the KM+-treated corneas 24 h after the injury. Growth factors and growth factors receptors are key regulators of development, homeostasis and corneal wound healing. Hepatocyte growth factor has been characterized as a heparin-binding paracrine mediator of stromal–epithelial interactions (Wilson et al. 1994b). HGF is secreted by fibroblasts to regulate the functions of epithelial cells in many organs (Nakamura et al. 1989; Matsumoto et al. 1991; Montesano et al. 1991; Rubin et al. 1991; Wilson et al. 1994a). Concerning the corneal epithelial, HGF modulates the cell proliferation, motility and differentiation (Wilson et al. 1993, Wilson et al. 1994a) and its expression is upregulated in rabbit keratocytes after corneal epithelium wounding (Li et al. 1996). The HGF receptor expression in corneal epithelium, keratocyte and endothelial cells (Wilson et al. 1993) is also upregulated after wounding, probably contributing to the corneal healing process (Weidner et al. 1991). The c-Met overexpression detected in the KM+-treated corneas might be also a result of neutrophils influx in the early stage of corneal repair.

Therefore, KM+ may accelerate corneal epithelial wound healing through a mechanism that involves neutrophil migration towards the cornea stroma during the first hours after injury. The resultant cell activation would be accompanied, in a later phase, by the overproduction of proteins that exert crucial role in the corneal wound healing. In addition, the observed acceleration of the epithelial wound healing indicates that KM+ may outline a novel therapeutic approach to restore quickly the corneal integrity after injury in situations of refractive surgery, contact lens wear, infections, chemical burns, etc., preventing an unfavourable outcome, like as secondary infections and visual loss.

Acknowledgments

The authors thank Auristella de Mello Martins and Sandra Maria de Oliveira Thomaz for the technical assistance. This research was supported by FAPESP, FAEPA and CNPq.

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