July 2002
Volume 43, Issue 7
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Immunology and Microbiology  |   July 2002
Regulation of Endotoxin-Induced Keratitis by PECAM-1, MIP-2, and Toll-like Receptor 4
Saloni Khatri; Jonathan H. Lass; Fred P. Heinzel; W. Matthew Petroll; John Gomez; Eugenia Diaconu; Carolyn M. Kalsow; Eric Pearlman
Author Affiliations
  • Saloni Khatri
    From the Departments of Medicine and
    Ophthalmology, Case Western Reserve University and the Research Institute of University Hospitals of Cleveland, Cleveland, Ohio; the
  • Jonathan H. Lass
    Ophthalmology, Case Western Reserve University and the Research Institute of University Hospitals of Cleveland, Cleveland, Ohio; the
  • Fred P. Heinzel
    From the Departments of Medicine and
  • W. Matthew Petroll
    Department of Ophthalmology, Southwestern Medical Center at Dallas, Dallas, Texas; and
  • John Gomez
    From the Departments of Medicine and
  • Eugenia Diaconu
    From the Departments of Medicine and
    Ophthalmology, Case Western Reserve University and the Research Institute of University Hospitals of Cleveland, Cleveland, Ohio; the
  • Carolyn M. Kalsow
    Ocular Research Services, Mendon, New York.
  • Eric Pearlman
    From the Departments of Medicine and
    Ophthalmology, Case Western Reserve University and the Research Institute of University Hospitals of Cleveland, Cleveland, Ohio; the
Investigative Ophthalmology & Visual Science July 2002, Vol.43, 2278-2284. doi:
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      Saloni Khatri, Jonathan H. Lass, Fred P. Heinzel, W. Matthew Petroll, John Gomez, Eugenia Diaconu, Carolyn M. Kalsow, Eric Pearlman; Regulation of Endotoxin-Induced Keratitis by PECAM-1, MIP-2, and Toll-like Receptor 4. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2278-2284.

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Abstract

purpose. Bacterial lipopolysaccharide (LPS, endotoxin) is a potent stimulator of inflammatory responses and is likely to contribute to microbial keratitis and to the pathogenesis of sterile corneal ulcers. The purpose of the present study was to identify specific mediators of endotoxin-induced keratitis.

methods. The corneal epithelium of BALB/c, C3H/HeJ, and C3H/HeN mice was abraded, and Pseudomonas aeruginosa endotoxin (10 μg in 1 μL) was added. Stromal thickness and haze were measured by in vivo scanning confocal microscopy, and neutrophil recruitment determined by immunohistochemistry.

results. Pseudomonas endotoxin induced a significant increase in stromal thickness and haze compared with untreated control corneas at each time point examined, and the severity coincided with neutrophil infiltration into the corneal stroma. Furthermore, systemic depletion of neutrophils completely abrogated endotoxin-induced increases in stromal thickness and haze, indicating an essential role for these cells. Expression of platelet endothelial cell adhesion molecule (PECAM)-1 on vascular endothelium and production of macrophage inflammatory protein (MIP)-2 in the corneal stroma were also significantly elevated after exposure to endotoxin, and antibody blockade inhibited neutrophil recruitment to the cornea and abrogated endotoxin-induced increases in stromal thickness and haze. In LPS-hyporesponsive C3H/HeJ mice, PECAM-1 and MIP-2 were not upregulated after exposure to endotoxin, and endotoxin-induced keratitis did not develop in these mice.

conclusions. These findings demonstrate that endotoxin-induced keratitis is regulated by toll-like receptor-4 (TLR4)-dependent expression of PECAM-1 and MIP-2, which are essential for recruitment of neutrophils to this site and for development of endotoxin-induced stromal disease.

Lipopolysaccharide (LPS, endotoxin), an abundant glycolipid in the outer membrane of Gram-negative bacteria, is composed of variable outer sugar chains attached to a highly conserved inner core lipid A. It is also a major virulence factor that can cause inflammatory responses in tissues, the consequences of which can be severe and even lethal. 1 2 3 Although an effective response has to be generated to combat infection by Gram-negative bacteria, the inflammation in response to bacterial endotoxin can cause systemic reactions, such as bacterial sepsis and uveitis. 4 5  
Microbial keratitis can cause severe pain and discomfort and can lead to permanent visual loss from scarring and, on occasion, perforation. 6 In developing countries, microbial keratitis is a major cause of blindness, often as a consequence of agricultural injury, and monocular blindness is estimated to affect millions of individuals worldwide. 7 8 In the United States, microbial keratitis is most frequently associated with complications resulting from contact lens wear. 9 Given that the incidence of microbial keratitis due to contact lenses is 25,000 to 30,000 cases annually in the United States and that the cost of medical treatment as a result of these cases is estimated at between $15 and $30 million, microbial keratitis due to contact lens wear has a considerable medical and economic impact. 
Keratitis can also develop in the absence of detectable live bacteria. These sterile infiltrates, which are often associated with contact lens wear, include contact lens-associated peripheral ulcers (CLPU) and contact lens-associated red eye (CLARE), which are painful conditions that can result in impaired visual function. 10 11 Although the etiology of contact lens-associated sterile infiltrates has not been fully characterized, the notion that bacterial products are involved seems reasonable, given that bacterial products can be released into the tears after lysis of bacteria and that the intense neutrophil infiltrate in corneal biopsy specimens of patients with sterile infiltrates is consistent with a response to bacteria. 10  
In the present study, we developed a murine model for sterile, subclinical corneal infiltrates, using bacterial endotoxin, and we identified essential mediators in development of stromal inflammation. 
Materials and Methods
Murine Model of Endotoxin-Induced Keratitis
Eight- to 12-week-old BALB/c mice and C3H/HeN mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C3H/HeJ mice were obtained from Harlan Laboratories (Indianapolis, IN). Mice were anesthetized by intraperitoneal injection of 0.4 mL of a 1.2% solution of 2,2,2-tribromoethanol (Aldrich Chemical Co., Milwaukee, WI) containing 2.5% 2-methyl-2-butanol (tertiary amyl alcohol; Aldrich) dissolved in distilled water. With a sterile 26-gauge needle, the central corneal epithelium of both eyes was scarified with two contiguous epithelial abrasions as described by Schultz et al. 12 13 Based on those studies, in which 1, 10, and 100 μg LPS was used on rabbit corneas, 12 13 we found that 10 μg/μL Pseudomonas aeruginosa endotoxin serotype 10 (99.7% purity; Sigma Chemical Co., St. Louis, MO) was optimal when applied topically to the scarified corneas (data not shown). Control corneas were scarified and exposed to endotoxin-free H2O (Sigma). All animals were treated in accordance with guidelines provided in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Confocal Microscopy Through-Focusing
In vivo scanning confocal microscopy (Tandem Scanning, Reston, VA) was performed with a confocal microscopy through-focusing (CMTF) program that enables precise thickness measurements of corneal layers and a quantitative estimate of corneal backscattering. 14 This measurement has been shown to correlate with clinical haze measurements. 15 Mice were anesthetized, and 1 drop of 2.5% hydroxypropyl methylcellulose was applied to the objective tip to eliminate the bright-field reflections, and the objective was brought in contact with the cornea. The gain was kept constant at 3.0 in all the readings and images, and the intensity profiles were obtained with a lens speed of 160 μm/sec and a sampling rate of 60 video fields per second. 
From each of these profiles, stromal thickness was calculated as the distance between the posterior epithelium and the peak of the endothelium. Baseline intensity was taken as the area posterior to the cornea, and intensity measurements were determined from the scattering of the signal in the corneal stroma. Stromal haze or backscattering was then defined and calculated as the intensity of the reflected light signal (compared with baseline) integrated over the stromal thickness. In inflamed corneas, increased stromal haze or backscattering is taken as a loss of corneal clarity. 
Depletion of Neutrophils In Vivo
To deplete neutrophils in vivo, mice were injected intraperitoneally (IP) with 500 μg/mL anti-GR1 rat mAb RB6 8C5 (PharMingen, San Diego, CA) 24 hours before corneal abrasion and exposure to endotoxin. This antibody reacts with the GR-1 antigen on the surface of neutrophils and eosinophils and is effective in depleting these cells in vivo. 16 17 Control animals were injected IP with 500 μg/mL normal rat IgG (Sigma). 
Histology and Immunohistochemistry
Mice were killed by cervical dislocation, and eyes were enucleated and either processed for routine histology and staining with hematoxylin and eosin by standard methods or snap frozen in liquid nitrogen. Frozen sections (5 μm) were fixed in 4% formaldehyde for 25 minutes, washed with 0.05 M Tris buffer, and incubated with proteinase K for 10 minutes at room temperature. After further washing, sections were incubated for 2 hours at room temperature with 8 μg/mL anti-neutrophil mAb NIMP R14 (obtained from Achim Hoerauf, Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany). Sections were washed and incubated with FITC-conjugated anti-rat antibody (Vector Laboratories Inc., Burlingame, CA) in a darkened humid chamber for 45 minutes. After a wash in Tris buffer, slides were air dried and mounted (Vectashield; Vector Laboratories Inc.). The number of neutrophils in 5-μm corneal sections was determined by fluorescence microscopy (Olympus Optical Co. Ltd., Tokyo, Japan). All sections were from the central region of the cornea, and cells were counted at 400× magnification from limbus to limbus. Cells were counted in at least two sections from each eye, and the average for each cornea was used for statistical analysis. 
Detection and Quantification of PECAM-1 Expression on Limbal Vessels
Eyes were snap frozen in liquid nitrogen and stored at −70°C, and 5-μm sections were air dried overnight and stored at −20°C. Sections were fixed for 10 minutes in −20°C acetone. Slides were air dried and rehydrated in PBS (pH 7.4). Rat mAb to platelet endothelial cell adhesion molecule (PECAM)-1 (MEC 13.3, PharMingen), was diluted 1:100 in PBS containing 1% fetal calf serum, and incubated for 2 hours at room temperature. FITC anti-rat IgG (H+L; Caltag Laboratories, South San Francisco, CA) diluted 1:100 was used as a secondary antibody and incubated for 45 minutes. Stained sections were washed in PBS, and mounting medium (Vectashield; Vector) was added to inhibit quenching. 
Images of limbal vessels were captured with a digital camera (model DC330; Page-MTI Inc., Michigan City, IN) and Scion Image Software (Version 1.62c; National Institutes of Health, Bethesda, ML, modified by Scion Corp.). To evaluate the relative fluorescence intensity, the mean brightness value of the green channel of three intensely stained areas of the vessel was determined using image-analysis software (Photoshop ver. 5.0; Adobe Systems Inc., San Jose, CA) with a set 400-pixel-square area, as described previously. 18 Four vessels were analyzed from each eye, the background reading in unstained areas of the cornea was subtracted from these values, and the mean ± SE of the fluorescence intensity for each vessel was estimated. 
Detection of MIP-2 by ELISA
To determine the concentration of macrophage inflammatory protein (MIP)-2 in murine corneas, animals were killed, and corneas were carefully dissected to avoid removing surrounding conjunctival tissue and underlying iris. Corneas were then placed in 400 μL RPMI, and sonicated 90 seconds at 50 cycles/sec (VibraCell; Sonics & Materials, Danbury, CT). The presence of MIP-2 in supernatants was detected by 2-site ELISA, according to the manufacturer’s directions (R&D Systems, Minneapolis, MN). The limit of detection for MIP-2 was 1.5 pg/mL. 
Subconjunctival Injection of Anti-PECAM-1 and Anti-MIP-2 Antibodies
Anti-PECAM-1 (25 μg; rat IgG2a, MEC 13.3; PharMingen, San Diego, CA) or anti-MIP-2 (25 μg; R&D Systems) in 5 μL was injected into the subconjunctival space of the right eye with a 33-gauge needle and syringe (Hamilton, Reno, NV), and 25 μg of control rat IgG (Sigma) was injected into the left subconjunctival space. For depletion of both mediators, mAbs were combined so that 25 μg anti-MIP-2 and 25 μg anti-PECAM-1 were injected in a total volume of 10 μL. Both corneas were then abraded and exposed to endotoxin, as described earlier. 
Statistics
Statistical significance was determined with an unpaired t-test (Prism; Graph Pad Software, San Diego, CA). P < 0.05 was considered to be significant. 
Results
Endotoxin-Induced Keratitis Measured by In Vivo Confocal Microscopy
To determine the effect of endotoxin exposure on normal corneal structure and function, corneas of BALB/c mice were gently abraded and exposed to 10 μg P. aeruginosa endotoxin in 1 μL endotoxin-free dH2O. Stromal haze and thickness were measured at various time points thereafter, using the CMTF program described by Li et al. 14  
Figure 1A shows representative CMTF images after exposure to dH2O or endotoxin, and Figure 1B shows profiles derived from these images. Corneas exposed only to dH2O, which were identical with naïve corneas (not shown), showed well-defined epithelial and endothelial cell layers, with no apparent stromal haze. The profiles show the increased light intensity of epithelium and endothelium as distinct peaks, with a low-intensity stromal layer. In marked contrast, exposure of the abraded corneal epithelium to endotoxin induced a significant increase in stromal thickness and haze. 
As shown in Figure 1C , stromal thickness and haze in endotoxin-treated corneas were significantly elevated as early as 6 hours, compared with corneas exposed to H2O, although the maximal response was at 24 hours. Values in control, abraded corneas exposed to endotoxin-free H2O were never significantly higher than those in corneas from naïve mice (data not shown). Together, these findings demonstrate that addition of endotoxin to the abraded cornea stimulates significant changes in normal corneal structure. 
Neutrophil Infiltration to the Corneal Stroma
To determine whether endotoxin-induced stromal abnormalities are due to inflammatory cell infiltration, corneas were examined by in vivo confocal microscopy and immunohistochemistry. 
Confocal images of the anterior stroma show a pronounced cellular infiltrate in endotoxin-treated corneas, but not control corneas (Fig. 2A) , and histologic and immunohistochemical analysis indicate that neutrophils comprise a major component of the infiltrate (Fig. 2B) . These cells were numerous in the perivascular region of the cornea, indicating that limbal vessels are a major source of neutrophils in the corneal stroma. Neutrophils were also found in close association with corneal endothelium. Figure 2C shows that neutrophils were recruited to the corneal stroma within 6 hours of exposure to endotoxin, with neutrophil numbers peaking at 24 hours, coincident with maximum stromal thickness and haze. 
Abrogation of Endotoxin-Induced Keratitis after Neutrophil Depletion
To determine the role of neutrophils in endotoxin-induced keratitis, mice were injected IP with anti-neutrophil (anti-GR1) antibody before endotoxin treatment. As shown in Figure 3 , these mice had significantly fewer neutrophils in the corneal stroma than did mice treated with control rat IgG. Consistent with this observation, stromal thickness and haze were not elevated in neutrophil-depleted mice after addition of endotoxin, indicating that endotoxin-induced keratitis is due to neutrophil infiltration of the corneal stroma. 
PECAM-1 Expression on Limbal Vessels
Previous studies have demonstrated a role for PECAM-1 in neutrophil recruitment into the cornea after infection with herpes simplex virus 19 and exposure to parasite antigens. 18 To determine whether PECAM-1 is upregulated in endotoxin-induced keratitis, corneas of BALB/c mice were abraded and exposed to H2O or endotoxin as before. Groups of mice were killed after 6 or 24 hours, and eyes were sectioned and stained with Ab to PECAM-1. As shown in Figure 4 , PECAM-1 expression on vascular endothelial cells in limbal blood vessels was significantly elevated at both time points after exposure to endotoxin compared with control corneas. 
MIP-2 Production in Endotoxin-Treated Corneas
In addition to elevated expression of vascular cell adhesion molecules, recruitment of inflammatory cells from blood vessels to tissues is also dependent on expression of chemotactic molecules. To determine whether MIP-2 is upregulated in endotoxin-induced keratitis, corneas were dissected 6 and 24 hours after endotoxin treatment, sonicated, and MIP-2 in the supernatant was measured by ELISA. 
As shown in Figure 5 , MIP-2 was elevated at 6 hours in endotoxin-treated corneas, but not in control corneas. However, MIP-2 was not detected in corneas removed 24 hours after exposure to endotoxin. Furthermore, separation of epithelium and stroma showed that MIP-2 was produced in the corneal stroma, rather than the epithelium (data not shown). 
Effect of Subconjunctival Injection of Antibody to PECAM-1 and MIP-2
To determine whether PECAM-1 and MIP-2 mediate neutrophil recruitment to the corneal stroma and development of endotoxin-induced keratitis, mice were injected into the subconjunctival space with either anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG, immediately before corneal abrasion and exposure to endotoxin. 
As shown in Figure 6 , neutrophil recruitment to the corneal stroma was significantly impaired after injection of anti-PECAM-1, anti-MIP-2, or both antibodies. Similarly, blockade of these mediators completely inhibited endotoxin-induced changes in stromal thickness and haze. 
Because blockade of either PECAM-1 or MIP-2 activity completely abrogated development of endotoxin-induced keratitis, these findings indicate that both mediators are essential for neutrophil recruitment to the cornea and for development of stromal abnormalities. 
Role of Toll-like Receptor-4 in Expression of PECAM-1 and MIP-2
Because toll-like receptor-4 (TLR4) activation is essential for endotoxin-induced inflammatory responses in mammalian cells, 20 we next examined the role of TLR4 in endotoxin-induced keratitis in C3H/HeJ mice. These mice have a single point mutation in the Tlr4 gene that renders them LPS hyporesponsive. 20 21 Corneas of C3H/HeJ and congenic (LPS responsive) C3H/HeN mice were abraded and exposed to endotoxin, and expression of PECAM-1 on limbal vessels and MIP-2 production in the corneas was evaluated. 
As shown in Figure 7 , expression of PECAM-1 and production of MIP-2 were significantly elevated in C3H/HeN mice compared with that in LPS hyporesponsive C3H/HeJmice, indicating that activation of TLR4 is essential for upregulation of these key mediators. 
Role of TLR4 in Endotoxin-Induced Keratitis
To determine whether TLR4 also regulates endotoxin-induced neutrophil infiltration and development of stromal edema and haze, corneas of C3H/HeJ and C3H/HeN were abraded and exposed to endotoxin, and neutrophil recruitment, stromal thickness, and stromal haze values were assessed. 
As shown in Figure 8 , corneas of C3H/HeN mice exposed to endotoxin showed pronounced neutrophil infiltration, resulting in increased stromal thickness and stromal haze. In marked contrast, corneas of C3H/HeJ mice exposed to endotoxin had significantly fewer neutrophils and significantly less stromal thickness and haze. Together, these findings demonstrate that activation of TLR4 is a critical step in development of endotoxin-induced keratitis. 
Discussion
In the mammalian cornea, disruption of normal function due to trauma, infection, or stromal and endothelial dystrophies result in loss of corneal transparency, impaired visual function and, in severe cases, blindness. Because the transparent nature of the mammalian cornea is maintained by a highly organized arrangement of collagen fibrils in addition to a tightly regulated level of hydration, 22 the physical presence of numerous infiltrating cells is likely to disrupt normal fibrillar arrangement, as well as corneal endothelial cell function, resulting in stromal edema and haze. 
In the present study, endotoxin-mediated loss of normal corneal function was indicated by increased stromal thickness and stromal haze that developed in the scarified corneal surface exposed to endotoxin. Endotoxin-induced keratitis was a direct result of extensive neutrophil infiltration to the corneal stroma, and recruitment of neutrophils to this site was mediated by PECAM-1 and MIP-2. Increased expression of PECAM-1 and MIP-2 and subsequent development of endotoxin-induced keratitis were shown to be regulated by TLR4. The latter observation is consistent with a recent report that functional TLR4 is expressed on corneal epithelial cells. 23  
Our findings on the role of neutrophils in keratitis are consistent with earlier reports in which rabbit corneas were injected with endotoxin, and neutrophils were found to disrupt normal corneal structure by inhibiting collagen synthesis. 24 In that study, ultrastructural analysis revealed the presence of vacuoles in the stroma, and the investigators suggested that structural damage is mediated by proteolytic enzymes such as collagenase and elastase that are released from neutrophil granules, 24 a notion supported by findings that intrastromal injection of neutrophil granules induce similar changes in collagen structure. 25  
Earlier studies also demonstrated that neutrophil depletion by whole-body irradiation or nitrogen mustard (which is likely to deplete cells other than neutrophils) inhibits Pseudomonas keratitis in guinea pigs 26 and endotoxin-induced keratitis in rabbits. 24 These investigators also concluded that neutrophil recruitment to the central cornea after exposure to Pseudomonas endotoxin or live bacteria was primarily from limbal vessels. 27  
Although the tear film could be a source of neutrophils in the corneal stroma, limbal and conjunctival blood vessels appear to be the primary source of these cells. Neutrophils were abundant in the perivascular region of limbal vessels as early as 6 hours after exposure to LPS. Extravasation of leukocytes from blood vessels into the tissue involves a complex series of events between leukocytes and vascular endothelial cells in which there is an initial selectin-mediated, low-affinity tethering and rolling. 28 If the inflammatory stimulus persists, higher-affinity binding is initiated that is mediated by integrins on the leukocytes and members of the immunoglobulin superfamily on vascular endothelial cells. PECAM-1 is expressed at highest levels at endothelial cell junctions and plays a critical role in transmigration of leukocytes across the endothelium and into the tissue, 29 30 31 32 binding primarily by homophilic interactions to PECAM-1 on the neutrophil surface. 33 34 Cells then migrate through the tissue, following a chemotactic gradient mediated at least in part by chemokines. 28  
In the present study, the CXC chemokine MIP-2, which is a murine functional homologue of IL-8, 35 36 showed a nonredundant role in neutrophil recruitment to the cornea in endotoxin-induced keratitis. This finding is consistent with previous reports from this laboratory and others in which blockade of MIP-2 receptor interactions inhibits neutrophil recruitment to the cornea in Pseudomonas keratitis, herpes simplex keratitis and Onchocerca volvulus keratitis. 37 38 39 In the present study, both PECAM-1 and MIP-2 appeared to be essential mediators of neutrophil recruitment to this site, because antibody to either molecule significantly inhibited neutrophil infiltration, and antibody to both mediators had no additional effect. 
Because endotoxin and live bacteria induce neutrophil infiltration by the same mediators, it is possible that endotoxin contributes to the pathogenesis of Pseudomonas keratitis by mediating recruitment of neutrophils through TLR4 signaling. Our studies, using a model for river blindness caused by the parasitic worm Onchocerca volvulus, also identified an important role for neutrophils and showed that neutrophil recruitment to the cornea is mediated by PECAM-1 and MIP-2. 18 37 40 The similarity of these findings with those in bacterial and endotoxin-induced keratitis appears to be due to the presence of endosymbiotic Wolbachia bacteria, which are abundant in these worms and are transmitted transovarially. 41 42 In that study, we found that neutrophil recruitment to the cornea and development of keratitis is almost entirely dependent on the presence of Wolbachia endobacteria in parasite extracts and TLR4 signaling. 43  
Although we have identified PECAM-1 and MIP-2 as important mediators of neutrophil recruitment to the cornea in endotoxin-induced keratitis, we cannot eliminate the possibility that other chemokines, such as MIP-1α, contribute to neutrophil recruitment, as indicated in herpes simplex keratitis, 44 or that other vascular adhesion cell molecules, such as CD18 and intercellular adhesion molecule (ICAM)-1 are involved. 13 45 However, given the observations from our laboratory and others, 18 19 38 39 it appears that PECAM-1 and MIP-2 are key mediators of neutrophil recruitment from limbal vessels to the corneal stroma. 
Results from the present study in LPS-hyporesponsive C3H/HeJ mice are similar to those found in the murine model of endotoxin-induced uveitis, in which these mice developed mild keratitis compared with that in C3H/HeN mice. 5 However, in that model, BALB/c mice had significantly less severe uveitis than did C3H/HeN mice, 5 whereas in the present study, we found no differences in severity of LPS-induced keratitis between the two strains. 
These findings also demonstrate that endotoxin-induced keratitis is regulated by TLR4, which is the major receptor for endotoxin on eukaryotic cells. 46 47 48 49 Signaling through this pathway, in the context of other receptors including CD14 and lipid A-binding protein, leads to nuclear factor (NF)-κB activation of genes encoding proinflammatory cytokines, such as IL-1 and TNF-α. 48 50 51 Because these proinflammatory cytokines stimulate keratocytes to produce IL-8 and gro-α, 52 53 and IL-1-β regulates production of MIP-2 in Pseudomonas keratitis, 54 it is likely that this pathway is also involved in endotoxin-induced keratitis. Similarly, IL-1α and TNF-α can regulate adhesion molecule expression, including PECAM-1, on vascular endothelial cells. 55 56  
Given these observations, the sequence of events leading to endotoxin-induced keratitis is likely to involve activation of TLR4 on resident corneal cells and secretion of proinflammatory cytokines, which then stimulate production of MIP-2 by keratocytes and expression of PECAM-1 on vascular endothelial cells. MIP-2 and PECAM-1 could mediate recruitment of neutrophils to the corneal stroma and thus induce stromal edema (possibly through endothelial dysfunction) and structural changes in corneal architecture. 
Further studies on the pathways leading to endotoxin-mediated stromal keratitis will identify TLR4-expressing cells in the cornea and characterize other mediators of inflammation that may be targeted for immune-based therapies. 
 Supported by a research grant from Bausch & Lomb, Inc. (EP), by National Eye Institute Grants EY10320 (EP) and EY11373 (JHL), by grants from the Research to Prevent Blindness Foundation and the Ohio Lions Research Foundation, and by the VA Medical Research Service (FPH) and by National Institute of Allergy and Infectious Disease Grants RO1AI35979 and AI45602 (FPH).
 Submitted for publication November 8, 2001; revised March 6, 2002; accepted April 1, 2002.
 Commercial relationships policy: E (CMK), F (all authors).
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
 Corresponding author: Eric Pearlman, Division of Geographic Medicine, Case Western Reserve University, School of Medicine, W137, 2109 Adelbert Road, Cleveland, OH 44106-4983; [email protected].
 
Figure 1.
Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
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Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
Figure 1.
 
Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
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Figure 2.
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
View OriginalDownload Slide
 
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
Figure 2.
 
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
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Figure 3.
Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
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Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
Figure 3.
 
Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
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Figure 4.
PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
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PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
Figure 4.
 
PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
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Figure 5.
MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
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MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
Figure 5.
 
MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
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Figure 6.
Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
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Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
Figure 6.
 
Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
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Figure 7.
PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
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PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
Figure 7.
 
PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
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Figure 8.
LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
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LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
Figure 8.
 
LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
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The authors thank Denise Hatala for expert technical support and Nathan Blackwell and Ravi Berger for many helpful discussions and critical reading of the manuscript. 
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Figure 1.
Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
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Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
Figure 1.
 
Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
Effect of LPS on stromal thickness and stromal haze. Corneal epithelium of BALB/c mice was abraded and 10 μg P. aeruginosa LPS or endotoxin-free H2O was added. Corneas were then examined by CMTF. (A) Representative two-dimensional CMTF images showing the corneal epithelium, stroma and endothelium 24 hours after exposure to LPS or dH2O. (B) Representative intensity profiles derived from these images. Peaks representing epithelial and endothelial layers of the cornea are indicated. Note the differences in thickness and area under the curve in the stromas of LPS and dH2O-treated corneas. (C) Stromal thickness and stromal haze measurements calculated from these profiles. Data are the mean ± SEM of measurements in five mice per group. Both parameters were significantly elevated after exposure to LPS (*P < 0.05). Mean (±SD) stromal thickness of naïve BALB/c mouse corneas was 67.24 ± 4.03 μm, and stromal haze was 1005.5 ± 201.7.
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Figure 2.
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
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Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
Figure 2.
 
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
Neutrophil infiltration of the corneal stroma after exposure to LPS. Corneas were abraded and exposed to LPS or H2O. After 24 hours, corneas were examined by CMTF, and eyes were removed, sectioned, and immunostained with antibody specific for murine neutrophils. (A) CMTF images of the anterior stroma showing few cells present (probably resident keratocytes) after exposure to H2O compared with a pronounced cellular infiltrate after exposure to LPS. (B) Limbal region of corneas exposed to LPS. Note the limbal vessel (large arrow) containing cells with polymorphonuclear morphology. These cells are also in the perivascular region and are associated with the corneal endothelium (small arrow). Inset: section immunostained with NIMP-14 antibody to detect neutrophils. Original magnification, ×400. (C) Temporal recruitment of neutrophils to the corneal stroma. The number of neutrophils per 5-μm section of the corneal stroma was determined by direct counting. Data are the mean ± SEM of measurements in five mice per group (*P < 0.05) between LPS- and H2O-treated corneas. The results are representative of three repeat experiments.
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Figure 3.
Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
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Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
Figure 3.
 
Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
Effect of neutrophil depletion on endotoxin-induced increases in stromal thickness and stromal haze. Mice were injected IP with either rat anti-mouse GR1 mAb to deplete neutrophils or with normal rat IgG before epithelial abrasion and addition of P. aeruginosa endotoxin. After 24 hours, stromal thickness and stromal haze were measured by CMTF, and the number of neutrophils in the stroma was determined by immunohistochemistry and direct counting. Note the decreased number of neutrophils in the corneal stroma after injection of anti-GR1 (RB6 8C5) compared with control antibody, and that stromal thickness and stromal haze were significantly diminished in the absence of neutrophils. Data points represent individual corneas.
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Figure 4.
PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
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PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
Figure 4.
 
PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
PECAM-1 expression on limbal vessels after exposure to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. After 24 hours, eyes were snap frozen, and 5-μm sections were immunostained with rat antibody to PECAM-1 and detected using FITC labeled anti-rat antibody. Relative fluorescence intensity was determined, and the mean ± SEM for six corneas per group are shown. Photomicrographs show representative vessels from corneas of BALB/c mice exposed to either H2O or LPS.
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Figure 5.
MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
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MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
Figure 5.
 
MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
MIP-2 production in corneas exposed to LPS. Corneas of BALB/c mice were abraded and exposed to LPS or H2O. Mice were killed after 6 or 24 hours, corneas were disrupted by sonication, and MIP-2 production was determined by ELISA. Data points are the mean ± SEM of measurements in six corneas per group.
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Figure 6.
Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
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Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
Figure 6.
 
Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
Effect of anti-PECAM-1 and anti-MIP-2 on LPS induced keratitis. BALB/c mice were given a single injection of 25 μg anti-PECAM-1, anti-MIP-2, both antibodies, or control rat IgG into the subconjunctival space before corneal abrasion and exposure to LPS. Data points represent individual corneas. (P < 0.001 for rat IgG compared with anti-PECAM-1, anti-MIP-2, and both antibodies for all parameters measured). The experiment was repeated twice with similar results.
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Figure 7.
PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
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PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
Figure 7.
 
PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
PECAM-1 expression and MIP-2 production in corneas of C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ mice (hyporesponsive to LPS) and congenic C3H/HeN mice (normal LPS responsive) were abraded and exposed to LPS as described in Figure 1 . MIP-2 production in the cornea was measured after 6 hours, and PECAM-1 expression on limbal vessels were determined after 6 and 24 hours. Both PECAM-1 and MIP-2 were elevated in C3H/HeN mice, but not in C3H/HeJ mice. Data are the mean ± SD of measurements in five mice per group and are representative of two repeat experiments.
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Figure 8.
LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
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LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
Figure 8.
 
LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
LPS-induced keratitis in C3H/HeJ and C3H/HeN mice. Corneas of C3H/HeJ and C3H/HeN mice were exposed to LPS as before. Twenty-four hours later, stromal thickness and stromal haze were measured by CMTF, and neutrophils were examined by immunohistochemistry. Note the diminished response for all parameters in C3H/HeJ mice compared with C3H/HeN mice. Data points represent individual corneas. The experiment was repeated twice with similar results.
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Copyright 2002 The Association for Research in Vision and Ophthalmology, Inc.
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