January 2000
Volume 41, Issue 1
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Immunology and Microbiology  |   January 2000
Adhesion Molecule Expression in a Rat Model of Staphylococcus aureus Endophthalmitis
Author Affiliations
  • Michael J. Giese
    From the Jules Stein Eye Institute and Departments of Ophthalmology, and
    Pathology and Laboratory Medicine, University of California Los Angeles School of Medicine.
  • David C. Shum
    From the Jules Stein Eye Institute and Departments of Ophthalmology, and
  • Sylvia A. Rayner
    From the Jules Stein Eye Institute and Departments of Ophthalmology, and
  • Bartly J. Mondino
    From the Jules Stein Eye Institute and Departments of Ophthalmology, and
  • Judith A. Berliner
    Pathology and Laboratory Medicine, University of California Los Angeles School of Medicine.
Investigative Ophthalmology & Visual Science January 2000, Vol.41, 145-153. doi:
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      Michael J. Giese, David C. Shum, Sylvia A. Rayner, Bartly J. Mondino, Judith A. Berliner; Adhesion Molecule Expression in a Rat Model of Staphylococcus aureus Endophthalmitis. Invest. Ophthalmol. Vis. Sci. 2000;41(1):145-153.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine whether Staphylococcus aureus and its components induce expression of E-selectin and intercellular adhesion molecule (ICAM)-1 in rat ocular tissues and on human endothelial cells in culture.

methods. Experimental and control rat eyes were injected with 80 colony-forming units of viable S. aureus and lipopolysaccharide-free sterile saline (NS), respectively. Eyes were enucleated and immediately frozen. E-selectin and ICAM-1 expression were evaluated on frozen sections by using standard immunohistochemical techniques. Using an enzyme-linked immunoassay, in vitro expression of E-selectin and ICAM-1 was evaluated on macrovascular endothelial cells after stimulation with S. aureus and selected purified components.

results. In S. aureus–injected eyes, E-selectin and ICAM-1 expression peaked at six to 24 hours, decreased slightly at 24 and 48 hours, and further declined by 72 hours. However, in NS-injected eyes, peak levels of E-selectin and ICAM-1 were seen at 6 hours, after which expression declined in the areas in which an increase was previously observed. In in vitro assays, peptidoglycan (0.01 μg/ml) induced a fourfold increase in E-selectin (P < 0.0001) and a twofold increase in ICAM-1 (P < 0.002) expression. Ribitol teichoic acid (RTA) (1 μg/ml) induced a twofold increase in E-selectin (P < 0.0001) and a threefold increase in ICAM-1 (P < 0.0001) expression.

conclusions. Eyes injected with S. aureus demonstrated a more intense and prolonged expression of both E-selectin and ICAM-1 than did eyes injected with NS. In addition, S. aureus components induced the in vitro expression of these adhesion molecules on macrovascular endothelial cells. The relevance of these findings to microvascular endothelial cells is yet to be determined.

Staphylococcus aureus is an important cause of postoperative endophthalmitis 1 2 3 and is associated with a rapid onset and a poor visual outcome. 4 5 6 7 8 Tissue destruction associated with endophthalmitis can be attributable to direct bacterial interactions and/or products and the host inflammatory response. Before bacterial killing and removal can occur, inflammatory cells must transmigrate into the site of infection. Infiltration of inflammatory cells is mediated by adhesion molecules expressed on the surface of both endothelial and inflammatory cells. 
The interaction of leukocytes with vascular endothelial cells represents a key control point in normal lymphocyte recirculation and leukocyte recruitment during inflammatory responses. The process of leukocyte recruitment into an area of inflammation follows a three-step paradigm. 9 It begins with the margination of inflammatory cells to the slower flow of the peripheral blood column. Leukocytes loosely adhere to endothelial cells, and that induces rolling of the inflammatory cell along the vessel’s luminal surface. This“ rolling” process is mediated by sialyated Lewis X moieties on leukocytes and by adhesion molecules called selectins on endothelial cells. 10 11 E-selectin is a major endothelial ligand mediating the entry of neutrophils, 12 the predominant infiltrating inflammatory cell population in acute bacterial infections. After binding to selectins, inflammatory cells sample the local environment, and if the appropriate signals are present, activation of the leukocyte occurs, resulting in firm adhesion to the endothelial cell. After this process, leukocytes transmigrate between endothelial cells into tissue. One important mediator of firm adhesion of leukocytes is intercellular adhesion molecule (ICAM)-1. It plays an important role in the regulation of adhesion and migration of all types of inflammatory cells. 13  
Expression of leukocyte adhesion molecules is mediated by a number of endothelial and leukocyte activators. Cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)-α are one class of endothelial activators. Bacterial components such as Gram-negative bacterial lipopolysaccharide (LPS) also are able to induce the expression of adhesion molecules. Little published information is available regarding Gram-positive bacterial components and their ability to activate endothelial cells and initiate inflammatory cell migration. 14 15 16 17 18  
The mechanisms of leukocyte infiltration into the eye during bacterial endophthalmitis are not well understood. Bacteria can actively grow in the vitreous and produce extracellular products that presumably interact with ocular tissues. The primary goal of these studies was to determine the type and time course of adhesion molecule expression after S. aureus injection in an established rat model of bacterial endophthalmitis. 19 Our secondary objective was to investigate the contribution of bacterial components such as cell wall (CW), its components ribitol teichoic acid (RTA) and peptidoglycan (PG), and the secreted product α-hemolysin to the induction of adhesion molecule expression on vascular endothelial cells in culture. Specifically, we examined the in vivo expression of E-selectin and ICAM-1 in ocular tissues after injection of S. aureus and the in vitro expression of adhesion molecules after stimulation with S. aureus components. 
Methods
Bacterial Strains and Components
In this study, a wild-type (WT) strain (RN6390) of S. aureus was used in the in vivo experiments. RN6390 is a laboratory strain that maintains its hemolytic pattern when propagated on sheep erythrocyte agar and produces α-, β-, and δ-hemolysin; lipases; and fibronectin-binding protein. 20 The WT and a mutant strain of the WT S. aureus strain, deficient in the production of hemolysins and lipase, 20 were used in our in vitro studies. Both strains of S. aureus were gifts from Ambrose Cheung, Rockefeller University, New York, New York. Isolates were maintained in sheep blood at −70°C. In preparation for use, bacteria were plated onto rabbit or sheep blood agar plates and incubated at 37°C for 24 hours. A discrete colony was subcultured into sterile tryptic soy broth (Sigma, St. Louis, MO) and incubated in a shaking water bath at 37°C for 18 hours. The bacterial culture was centrifuged at 1900g for 10 minutes and the pellet washed with 0.85% LPS-free sterile saline (NS) and centrifuged, and the resultant pellet was resuspended in NS. A dilution was made to a spectrophotometric optical density (Spectronic 21; Bausch and Lomb, Rochester, NY) of 0.19 to 0.20 at 530 nm, which corresponded to a viable bacterial count of approximately 1.5 × 108 colony-forming units (CFU) per milliliter. This suspension of S. aureus was adjusted by serial dilution with NS to yield a final concentration of approximately 80 CFU per 25μ l for intravitreal injection. S. aureus was heat-killed by heating the final concentration at 80°C for 30 minutes. 
Purified S. aureus RTA was purchased from Meridian Diagnostics (Cincinnati, OH) and S. aureus α-hemolysin was purchased from Toxin Technology (Sarasota, FL). CW was purified from an ocular isolate of S. aureus by a previously described method. 21 Ribitol teichoic acid, α-hemolysin, and CW were tested for the presence of LPS with the limulus amebocyte lysate assay and were found to be below detectable limits (0.002 μg/μl). 
Intravitreal Injections
Twenty-seven female Lewis rats, 8 to 10 weeks old (Harlan Sprague–Dawley, San Diego, CA) raised in a pathogen-free environment were used in this study. Studies were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Under direct observation, the right eye of each experimental rat received a 25-μl intravitreal injection of either S. aureus or NS, following a procedure previously described. 19 Before the intravitreal injection, paracentesis was performed 1 mm anterior to the limbus to limit extrusion of the inoculum. Additionally, 25 μl of the S. aureus suspension was plated onto rabbit and sheep blood agar plates and incubated at 37°C for 24 hours to confirm the approximate number of S. aureus injected into the eye. Rats were killed 6, 24, 48, and 72 hours after injections. 
In Vivo Adhesion Molecule Expression
Eyes were sectioned on a cryostat (Reichert–Jung 2800 Frigocut E; Leica, Deerfield, IL) at 6 to 8 μm and stored at −70°C. For both ICAM-1 and E-selectin staining, tissue sections were air dried and fixed in ice-cold 95% methanol for 5 minutes. After fixation, sections were washed with 1× phosphate-buffered saline (PBS, pH 7.4). Endogenous peroxidase was blocked for 20 minutes at room temperature (RT) with 0.6% hydrogen peroxide. For E-selectin detection, an avidin-biotin block (Vector, Burlingame, CA) was performed followed by a wash with PBS. Tissue sections in the E-selectin and ICAM-1 groups were then blocked with PBS containing 10% horse serum (Gibco, Grand Island, NY) and 1% bovine serum albumin (BSA; Sigma) for 1 hour at RT. For ICAM-1, mouse anti-rat ICAM antibody (Ab; clone 1A29), 1 to 5μ g/ml (Serotec, Raleigh, NC) and for E-selectin, mouse anti-rabbit E-selectin, 1 μg/ml (monoclonal [m]Ab 14G2, a gift from Barry A. Wolitsky, Hoffman–LaRoche, Nutley, New Jersey), which cross-reacts with rat E-selectin 22 were applied to the tissue. Control tissues were incubated with a mouse immunoglobulin (Ig)G isotype control Ab (Dako, Carpinteria, CA) overnight at 4°C. After incubation with primary Ab, a biotinylated horse anti-mouse IgG (1 μg/ml; Vector) was applied to the tissue and incubated at RT for 30 minutes. E-selectin and ICAM-1 were detected using the avidin-biotin complex (Vector). ICAM-1 was developed with 3-amino-9-ethylcarbazole (Sigma) producing red staining and E-selectin was developed with 3,3′-diaminobenzidine tetrahydrochloride (Sigma) producing brown staining. Tissues were counter-stained with hematoxylin (Biømeda, Foster City, CA). 
The intensity of staining in the iris, ciliary body (CB), retina, and choroid, and the approximate area of tissue stained per section were scored on a scale of 0 (absent) to 3 (strong) by two observers in a masked fashion. Random sections from noninjected, NS and S. aureus–injected eyes of three rats (n = 3) were stained in triplicate, and mean levels of staining intensity were calculated for each time point. 
In Vitro Adhesion Molecule Assay
Aortic endothelial cells were obtained from human donor tissue according to a previously described procedure. 23 Fifth- to eighth-passage cells were plated onto 96-well plates (Costar, Cambridge, MA) and grown to confluence. Endothelial cells in Medium 199–5% fetal bovine serum were stimulated with live S. aureus strains, heat-killed S. aureus, CW, PG, RTA, andα -hemolysin. To detect expression of E-selectin, cells were washed and immediately fixed with 1% paraformaldehyde (PF) on ice for 20 minutes. After fixation, cells were washed with 1× tris-buffered saline (TBS)-5% glycine and then 1× PBS. Next, mouse anti-human E-selectin mAb (Biosource, Camarillo, CA) was added (1 μg/ml) and incubated overnight at 4°C. For ICAM, cells were incubated with a mouse anti-human ICAM mAb (1 μg/ml; Biosource) on ice for 1 hour. After incubation, cells were washed and fixed with 1% PF on ice for 20 minutes. After fixation, cells were washed with 1× TBS-5% glycine then with 1× PBS-1% BSA. After incubation, a goat anti-mouse horseradish peroxidase–conjugated secondary Ab (Jackson ImmunoResearch, West Grove, PA) was added to the wells and incubated for 2 hours at RT. Cells were washed, and adhesion molecule expression was detected with o-phenylenediamine (Sigma). After 5 minutes, the reaction was stopped with 3 N hydrochloric acid, and the plates were read at 490 nm with a microplate reader (Thermo-max, Molecular Devices, Sunnyside, CA). Lipopolysaccharide (0.002 μg/ml) served as the positive control in all experiments. 
Statistical Analysis
For in vitro studies, each condition was tested in quadruplicate and experiments were repeated two to three times. For whole-cell enzyme-linked immunosorbent assays, analysis of variance with Fisher–Tukey least-significant difference criterion was used to analyze the data (Statview; Abacus Concepts, Berkeley, CA). Significance was determined at P < 0.05. 
Results
In Vivo E-Selectin and ICAM-1 Expression
All experimental animals used for the in vivo detection of E-selectin and ICAM-1 demonstrated clinical detectable inflammation after intravitreal injection of live S. aureus. Clinical inflammation peaked at approximately 24 hours and then declined. Minimal clinical inflammation was detected in NS-injected eyes. Very low levels of E-selectin were detected in ocular tissues of noninjected eyes (Table 1 ; Fig. 1L ). At 6 hours, moderate to strong degrees of CB and iris vessel staining were detected in eyes injected with S. aureus (Figs. 1A 1B ; Fig. 2 ) and NS (Figs. 1E 1F) whereas mild degrees of staining were seen in the choroid and retina (Fig. 1J) . Interestingly, retinal vessels of the S. aureus–injected eyes demonstrated a mild degree of staining that was located adjacent to the optic nerve head. This staining was localized to the outer plexiform and ganglion cell layers of the retina. At 24 hours, E-selectin expression decreased in the CB, iris (Figs. 1C 1D) and retina (Fig. 1K) of S. aureus–injected eyes but increased in the choroid. By 48 hours, E-selectin staining intensity further decreased in the CB, iris (Figs. 1H 1I) , choroid and retina of S. aureus–injected eyes. By 72 hours, staining decreased in all tissues and was similar to that in the NS-injected group. In NS-injected eyes, E-selectin expression decreased from the 6-hour peak levels (Figs. 1E 1F) to levels slightly higher than constitutive levels at 24 (Fig. 1G) , 48, and 72 hours. 
A weak degree of ICAM-1 staining in the iris, CB, and choroid was demonstrated in noninjected eyes (Table 2 ; Figs. 1M 1P ). This level was considered to be constitutive. At 6 hours, ICAM-1 staining intensity increased from the constitutive level to mild levels in the CB, iris, and choroid of S. aureus– and NS-injected eyes (Fig. 3) . A slight decrease in staining intensity was noted in NS-injected eyes at 24 hours, and the staining intensity remained at this level at all subsequent time points. The intensity of ICAM-1 staining at 24 hours increased from the 6-hour levels to moderate levels in the CB, iris, choroid, and retina of S. aureus–injected eyes (Figs. 1N 1O 1Q) . This staining was localized to the vessels of the anterior surface of the iris and to the nonpigmented ciliary epithelium and vessels of the CB. At 24 hours, moderate staining of retinal vessels in the outer plexiform and ganglion cells layers (Fig. 1Q) was seen. Staining of vessels of the optic nerve head also was detected at this time in S. aureus–injected eyes (Fig. 1R) but not in NS-injected eyes. In S. aureus–injected eyes, decreases in staining intensity were noted in the CB, choroid, and retina at 48 hours (data not shown), whereas staining in the iris remained at the 24-hour level. At 72 hours, ICAM-1 staining intensity in all ocular tissues of S. aureus–injected eyes decreased from the 48-hour level but remained higher than constitutive levels. 
In Vitro E-Selectin and ICAM-1 Expression
In initial studies, we stimulated endothelial cells for both 4 and 24 hours. At 24 hours there was no significant increase of adhesion molecule expression beyond that seen at 4 hours. Therefore, the remainder of the studies were performed at 4 hours. E-selectin expression was induced after stimulation with RTA and PG after 4 hours (Fig. 4) but was not detected with CW (data not shown). A statistically significant increase in E-selectin expression over control levels was detected with RTA (P < 0.02) and PG (P < 0.0005) at all tested concentrations. 
ICAM-1 was induced with PG and RTA but not with CW after stimulation for 4 hours (Fig. 5) . RTA induced statistically significant increases in ICAM-1 expression with 10- and 1-μg/ml concentrations (P < 0.0001). Borderline significant expression was seen with RTA at 0.1 μg/ml (P = 0.058). PG induced a statistically significant increase in ICAM-1 expression at all concentrations (0.1 μg/ml = P < 0.04). No significant consistent expression of E-selectin or ICAM-1 was observed below this concentration. 
Because of cell detachment caused by hemolysins, we were unable to detect expression of E-selectin and ICAM-1 after stimulation with live S. aureus. However, when endothelial cells were stimulated with a live mutant strain of S. aureus, without hemolysins and lipase, 20 at concentrations between 1.5 × 102 and 1.5 × 108 CFU, E-selectin and ICAM were induced (data not presented). Purifiedα -hemolysin did not induce expression of E-selectin or ICAM-1, nor did heat-killed WT S. aureus at concentrations of 1.5 × 102 to 1.5 × 106 CFU. However, heat-killed S. aureus at a concentration of 1.5 × 108 CFU induced expression of E-selectin (P < 0.01). Interestingly, heat-killed Escherichia coli at concentrations of 1.5 × 104 and 1.5 × 106 CFU induced expression of both E-selectin and ICAM-1 (data not shown). 
Discussion
Endophthalmitis is a disease characterized by the infiltration of inflammatory cells into the anterior and vitreous chambers of the eye. We have shown in separate studies that the intraocular inflammatory cellular response to the injection of viable S. aureus in the rat eye reaches a maximum at approximately 24 to 48 hours and then declines. 24 25 In addition, we have characterized this response and found it to be initially dominated by neutrophils with increasing recruitment of monocytes-macrophages, and lymphocytes at later times. Infiltration of all inflammatory cells is dependent on the expression of endothelial cell adhesion molecules such as E-selectin, ICAM, and vascular cell adhesion molecule (VCAM). Previous studies have shown that ICAM-1 is present at low levels on normal ocular tissues 26 27 28 29 30 31 32 and that all these molecules are increased in ocular disease states. 32 33 34 35 36 37 38 39 40 41  
Results from the present studies show that adhesion molecules were expressed in ocular tissues after intravitreal injection of viable S. aureus. Adhesion molecule expression precedes leukocyte entry, as published in our previous model. 24 Our in vivo experiments showed that E-selectin expression peaked at 6 to 24 hours in S. aureus–injected eyes, slowly declined, and returned to constitutive levels at 72 hours. The rapid and sustained expression of E-selectin in the iris, CB, and retinal vessels is similar to the in vivo findings of Suzuma et al. 42 in a rat model of endotoxin-induced uveitis. In this model, E-selectin expression was not detected in the iris, CB, or retina 5 hours after LPS injection but was detectable in these structures after 7 hours. Expression remained present in these tissues for 24 hours. Our findings also are comparable with other in vivo reports in which E-selectin expression was detectable on microvessels up to 72 hours after LPS injection in cutaneous models of inflammation. 43 The time course of E-selectin expression seen in our model is similar to that seen in in vitro studies of microvascular endothelial cells 44 45 but not large-vessel endothelial cells in which E-selectin is rapidly induced and rapidly decreased. 46 In vitro studies have demonstrated that dermal microvascular endothelial cells show a peak E-selectin expression at 6 to 8 hours and sustained expression after treatment with TNF. 44 In contrast to E-selectin, in vitro ICAM-1 expression peaks between 16 to 24 hours and persists at peak levels as long as proinflammatory cytokines are present. 47 Our in vivo ICAM-1 data are consistent with these findings. Upregulation of ICAM-1 expression was first seen at 6 hours, with maximal expression occurring at 24 hours followed by decreases in staining intensities at 48 and 72 hours. The sustained in vivo expression pattern of E-selectin and ICAM-1 seen in our model may be in response to vitreous bacterial products and/or cytokines whose production we have shown to be increased after injection of viable S. aureus. 25 In the present study, the 24-hour peak in ICAM-1 expression corresponded to the peak levels of intravitreal TNF-α, IL-1β, cytokine-induced neutrophil chemoattractant (CINC), and interferon-γ. 
Inflammatory cells can enter the eye from the CB, iris, choroid, and retinal vasculature. Our previous study 24 showed that most inflammatory cells entered the eye from the CB and iris, with fewer entering from the choroid and retina. The in vivo data presented in this study support this finding, because the CB and iris tissues demonstrated the most widespread and intense E-selectin and ICAM-1 staining pattern. This agrees with Bamforth et al., 48 who showed that the origin of leukocytes in the vitreous, after intravitreal IL-1β injection, was thought to be derived from the CB. Vessels of the CB are fenestrated and are not part of the blood–retinal barrier. Therefore, they may demonstrate an adhesion molecule expression and leukocyte transmigration pattern similar to that seen in other postcapillary endothelial cells but different from vessels that are part of the blood–retinal barrier. In our past work 24 and this present study, there was a small but significant increase in inflammatory cells and adhesion molecule expression associated with the retinal vessels of the optic nerve, suggesting that retinal vessels may be a source of vitreous cells. Bamforth et al., 48 have shown that leukocyte infiltration into the retina occurs from the retinal vasculature and that leukocytes may migrate through retinal vessel tight junctions or through the endothelial cell itself in response to intravitreal IL-1β injection. These same investigators have also shown that intravitreal injection of IL-1β and TNF-α lead to increased permeability of the blood–retinal barrier and infiltration of inflammatory cells. 49 50 The different staining intensities between the CB and retinal vessels may be due to different endothelial responses to host factors, Gram-positive bacteria and/or components. In vitro studies have shown that lymphocyte adhesion to brain and retinal endothelial cells differs from lymphocyte adhesion to endothelial cells of non–central nervous system origin. 51  
In vitro cell culture assays were used to determine whether S. aureus components may be responsible for induction of the adhesion molecules detected in ocular tissues. In our in vivo studies there were increases in both E-selectin and ICAM-1 expression at 6 hours; therefore, we chose to investigate the early induction phases of the response in vitro. To the best of our knowledge, this is the first report to demonstrate the ability of S. aureus RTA and PG to induce the in vitro expression of E-selectin and ICAM-1 on human aortic endothelial cells. In our studies, RTA generally was not as effective as PG and LPS in inducing E-selectin and ICAM-1 expression. Lower concentrations of PG induced the expression of adhesion molecules, but the concentration of PG needed to induce expression of E-selectin and ICAM-1 was still five times (0.01 μg/ml) greater than the minimal LPS concentration required for adhesion molecule expression (0.002 μg/ml). In studies by other investigators, it also has been demonstrated that greater concentrations of PG are needed to induce the production of cytokines from monocytes compared with LPS. 52 53 Interestingly, studies have shown that when PG is bound to lipoteichoic acid (LTA) it is a more potent stimulator of IL-1 than is LPS 54 and is associated with systemic inflammation. 55  
We were unable to detect expression of adhesion molecules after stimulation with S. aureus CW. The CW is composed of many products, including PG and RTA covalently linked. The inability of CW to induce expression may be because the CW preparation was not fragmented enough to allow exposure of the activating determinants. The concentrations of PG and RTA used in our study are consistent with those used in Kawamura et al. 15 who showed that S. aureus LTA induces the in vitro expression of E-selectin, ICAM-1, and VCAM. In their study, maximum E-selectin expression occurred with a LTA concentration of 10 μg/ml, which was 100 times greater than the amount of LPS needed to induce maximal E-selectin expression. The receptors for PG and RTA are not known, but it has been shown that a soluble form of PG binds to CD14 on monocytes inducing the release of IL-6. 56 Additionally, we were unable to demonstrate consistent expression of E-selectin and ICAM-1 after stimulation with α-hemolysin. We used concentrations as low as 0.001μ g/ml and did not observe any loss of endothelial cells (data not shown). 
In our in vitro system, heat-killed S. aureus at concentrations of 1.5 × 106 CFU/ml and less were unable to stimulate the expression of E-selectin and ICAM-1, but heat-killed E. coli induced expression of these molecules. The inability of heat-killed S. aureus to induce expression of adhesion molecules is consistent with our data showing that purified CW did not induce adhesion molecule expression. This finding is also consistent with a report by Noel et al. 16 in which heat-killed S. aureus failed to induce expression of E-selectin. Heat killing may alter the structure of the CW and the PG–RTA complex making it unable to directly activate endothelial cells to express adhesion molecules. Alternately, the component(s) responsible for activation of endothelial cells may be buried in the CW of S. aureus, whereas in E. coli the LPS molecules may be exposed on the outer aspect of the cell membrane, thus allowing for activation. We used macrovascular endothelial cells to evaluate responses to S. aureus and its components in vitro. The relevance of these findings to microvascular endothelial cells must be determined in future studies. However, the kinetics of induction of adhesion molecules in our in vitro studies were quite similar to the in vivo findings. 
In summary, our studies have suggested a role for E-selectin and ICAM-1 in leukocyte entry in S. aureus endophthalmitis. In addition, we have shown that Gram-positive bacterial products are able to induce the in vitro expression of E-selectin and ICAM-1. Our previous studies also suggested a role for cytokines in this process. Therefore, adhesion molecule expression by ocular tissues may be mediated directly by bacterial products or through intermediates such as cytokines. To achieve activation of the basal aspect of the endothelial cells in the iris, CB, and retinal vessels, bacterial products would have to diffuse through the vitreous, retina, and CB. This activation would result in the subsequent expression of adhesion molecules followed by the infiltration of leukocytes. Thus, our studies suggest that a combination of bacterial and host factors regulate the early stages of endophthalmitis and consequently may serve as therapeutic targets. 
 
Table 1.
 
E-selectin Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Table 1.
 
E-selectin Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Noninjected 6 Hours 24 Hours 48 Hours 72 Hours
NS WT NS WT NS WT NS WT
CB 0.05 ± 0.15 2.33 ± 1.0 2.14 ± 0.75 0.25 ± 0.54 1.33 ± 0.99 0.21 ± 0.35 0.70 ± 0.68 0.38 ± 0.32 0.29 ± 0.37
Iris 0.02 ± 0.08 2.58 ± 1.0 2.46 ± 0.63 0.38 ± 0.49 1.73 ± 0.89 0.64 ± 0.64 1.10 ± 0.74 0.28 ± 0.25 0.18 ± 0.31
Choroid 0.05 ± 0.15 0.81 ± 0.65 1.04 ± 0.48 0.25 ± 0.43 1.25 ± 0.88 0.46 ± 0.49 0.65 ± 0.41 0.20 ± 0.35 0.11 ± 0.28
Retinal vessels 0.00 0.42 ± 0.52 1.32 ± 0.64 0.06 ± 0.17 1.28 ± 1.0 0.05 ± 0.15 0.80 ± 0.63 0.05 ± 0.16 0.00
Figure 1.
 
Panel 1: E-selectin staining of ocular tissues. (A) Iris 6 hours after injection of WT S. aureus; (B) CB 6 hours after injection with WT S. aureus; (C) iris 24 hours after injection with WT S. aureus; (D) CB 24 hours after injection with WT S. aureus; (E) iris 6 hours after injection with NS; (F) CB 6 hours after injection with NS; (G) CB 24 hours after injection with NS; (H) iris 48 hours after injection with WT S. aureus; (I) CB 48 hours after injection with WT S. aureus; (J) E-selectin staining of a retinal vessel in the ganglion cell layer (arrow) and choroid (arrowhead) 6 hours after injection with WT S. aureus; (K) E-selectin staining of a retinal vessel in the outer plexiform layer (OPL; arrow) 24 hours after injection with WT S. aureus; and (L) noninjected eye with no E-selectin expression seen in the CB. Panel 2: ICAM-1 staining of ocular tissues: (M) ICAM-1 constitutive staining of CB from a noninjected eye; (N) ICAM-1 staining of CB 24 hours after injection with WT S. aureus. Note staining of the ciliary epithelium and choroid; (O) iris 24 hours after the injection of WT S. aureus; (P) constitutive staining of the choroid from a noninjected eye; (Q) staining of choroidal and vessels in the OPL (long arrow) 24 hours after injection with WT S. aureus; and (R) ICAM-1 staining of vessels of the optic nerve 24 hours after injection of WT S. aureus. Magnification, (A, C, and Q) ×675; (B) ×550; (D, I) ×600; (E, R) ×500; (F)× 620; (G, H, L, and P)× 625; (J) ×700; (K) ×850; (M) ×440; (N) ×200; (O) ×450.
Figure 1.
 
Panel 1: E-selectin staining of ocular tissues. (A) Iris 6 hours after injection of WT S. aureus; (B) CB 6 hours after injection with WT S. aureus; (C) iris 24 hours after injection with WT S. aureus; (D) CB 24 hours after injection with WT S. aureus; (E) iris 6 hours after injection with NS; (F) CB 6 hours after injection with NS; (G) CB 24 hours after injection with NS; (H) iris 48 hours after injection with WT S. aureus; (I) CB 48 hours after injection with WT S. aureus; (J) E-selectin staining of a retinal vessel in the ganglion cell layer (arrow) and choroid (arrowhead) 6 hours after injection with WT S. aureus; (K) E-selectin staining of a retinal vessel in the outer plexiform layer (OPL; arrow) 24 hours after injection with WT S. aureus; and (L) noninjected eye with no E-selectin expression seen in the CB. Panel 2: ICAM-1 staining of ocular tissues: (M) ICAM-1 constitutive staining of CB from a noninjected eye; (N) ICAM-1 staining of CB 24 hours after injection with WT S. aureus. Note staining of the ciliary epithelium and choroid; (O) iris 24 hours after the injection of WT S. aureus; (P) constitutive staining of the choroid from a noninjected eye; (Q) staining of choroidal and vessels in the OPL (long arrow) 24 hours after injection with WT S. aureus; and (R) ICAM-1 staining of vessels of the optic nerve 24 hours after injection of WT S. aureus. Magnification, (A, C, and Q) ×675; (B) ×550; (D, I) ×600; (E, R) ×500; (F)× 620; (G, H, L, and P)× 625; (J) ×700; (K) ×850; (M) ×440; (N) ×200; (O) ×450.
Figure 2.
 
Mean E-selectin staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 1 ).
Figure 2.
 
Mean E-selectin staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 1 ).
Table 2.
 
ICAM-1 Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Table 2.
 
ICAM-1 Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Noninjected 6 Hours 24 Hours 48 Hours 72 Hours
NS WT NS WT NS WT NS WT
CB 0.55 ± 0.27 1.18 ± 0.63 1.31 ± 0.68 1.00 ± 0.47 2.32 ± 0.78 1.08 ± 0.62 2.06 ± 0.73 1.06 ± 0.59 1.36 ± 0.98
Iris 0.77 ± 0.52 1.46 ± 0.65 1.61 ± 0.65 1.20 ± 0.42 2.21 ± 0.94 1.06 ± 0.58 2.19 ± 0.57 1.23 ± 0.61 1.31 ± 0.65
Choroid 0.73 ± 0.45 1.46 ± 0.50 1.06 ± 0.53 0.95 ± 0.46 2.00 ± 0.60 1.17 ± 0.53 1.28 ± 0.86 1.27 ± 0.73 1.06 ± 0.68
Retinal vessels 0.00 0.00 0.06 ± 0.17 0.10 ± 0.32 1.65 ± 0.63 0.23 ± 0.44 0.75 ± 0.65 0.11 ± 0.18 0.13 ± 0.35
Figure 3.
 
Mean ICAM staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 2 ).
Figure 3.
 
Mean ICAM staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 2 ).
Figure 4.
 
In vitro E-selectin expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in E-selectin expression are noted for all concentrations of RTA (10 and 1 μg/ml, P < 0.001; 0.1 μg/ml, P < 0.02). (B) Statistically significant increases in E-selectin expression are noted for all concentrations of PG (P < 0.0005). Data presented are representative samples of at least triplicate trials.
Figure 4.
 
In vitro E-selectin expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in E-selectin expression are noted for all concentrations of RTA (10 and 1 μg/ml, P < 0.001; 0.1 μg/ml, P < 0.02). (B) Statistically significant increases in E-selectin expression are noted for all concentrations of PG (P < 0.0005). Data presented are representative samples of at least triplicate trials.
Figure 5.
 
In vitro ICAM-1 expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in ICAM-1 expression were noted for RTA concentrations of 10 and 1 μg/ml (P < 0.0001) and a nearly significant increase with 0.1 μg/ml (P = 0.058). (B) Statistically significant increases in ICAM-1 expression were noted for PG concentrations of 10, 1, and 0.01 μg/ml (P < 0.0005) and 0.1 μg/ml (P < 0.04). Data presented are representative samples of at least triplicate trials.
Figure 5.
 
In vitro ICAM-1 expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in ICAM-1 expression were noted for RTA concentrations of 10 and 1 μg/ml (P < 0.0001) and a nearly significant increase with 0.1 μg/ml (P = 0.058). (B) Statistically significant increases in ICAM-1 expression were noted for PG concentrations of 10, 1, and 0.01 μg/ml (P < 0.0005) and 0.1 μg/ml (P < 0.04). Data presented are representative samples of at least triplicate trials.
The authors thank Yoon Cho and Mabel Pang for excellent technical assistance. 
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Figure 1.
 
Panel 1: E-selectin staining of ocular tissues. (A) Iris 6 hours after injection of WT S. aureus; (B) CB 6 hours after injection with WT S. aureus; (C) iris 24 hours after injection with WT S. aureus; (D) CB 24 hours after injection with WT S. aureus; (E) iris 6 hours after injection with NS; (F) CB 6 hours after injection with NS; (G) CB 24 hours after injection with NS; (H) iris 48 hours after injection with WT S. aureus; (I) CB 48 hours after injection with WT S. aureus; (J) E-selectin staining of a retinal vessel in the ganglion cell layer (arrow) and choroid (arrowhead) 6 hours after injection with WT S. aureus; (K) E-selectin staining of a retinal vessel in the outer plexiform layer (OPL; arrow) 24 hours after injection with WT S. aureus; and (L) noninjected eye with no E-selectin expression seen in the CB. Panel 2: ICAM-1 staining of ocular tissues: (M) ICAM-1 constitutive staining of CB from a noninjected eye; (N) ICAM-1 staining of CB 24 hours after injection with WT S. aureus. Note staining of the ciliary epithelium and choroid; (O) iris 24 hours after the injection of WT S. aureus; (P) constitutive staining of the choroid from a noninjected eye; (Q) staining of choroidal and vessels in the OPL (long arrow) 24 hours after injection with WT S. aureus; and (R) ICAM-1 staining of vessels of the optic nerve 24 hours after injection of WT S. aureus. Magnification, (A, C, and Q) ×675; (B) ×550; (D, I) ×600; (E, R) ×500; (F)× 620; (G, H, L, and P)× 625; (J) ×700; (K) ×850; (M) ×440; (N) ×200; (O) ×450.
Figure 1.
 
Panel 1: E-selectin staining of ocular tissues. (A) Iris 6 hours after injection of WT S. aureus; (B) CB 6 hours after injection with WT S. aureus; (C) iris 24 hours after injection with WT S. aureus; (D) CB 24 hours after injection with WT S. aureus; (E) iris 6 hours after injection with NS; (F) CB 6 hours after injection with NS; (G) CB 24 hours after injection with NS; (H) iris 48 hours after injection with WT S. aureus; (I) CB 48 hours after injection with WT S. aureus; (J) E-selectin staining of a retinal vessel in the ganglion cell layer (arrow) and choroid (arrowhead) 6 hours after injection with WT S. aureus; (K) E-selectin staining of a retinal vessel in the outer plexiform layer (OPL; arrow) 24 hours after injection with WT S. aureus; and (L) noninjected eye with no E-selectin expression seen in the CB. Panel 2: ICAM-1 staining of ocular tissues: (M) ICAM-1 constitutive staining of CB from a noninjected eye; (N) ICAM-1 staining of CB 24 hours after injection with WT S. aureus. Note staining of the ciliary epithelium and choroid; (O) iris 24 hours after the injection of WT S. aureus; (P) constitutive staining of the choroid from a noninjected eye; (Q) staining of choroidal and vessels in the OPL (long arrow) 24 hours after injection with WT S. aureus; and (R) ICAM-1 staining of vessels of the optic nerve 24 hours after injection of WT S. aureus. Magnification, (A, C, and Q) ×675; (B) ×550; (D, I) ×600; (E, R) ×500; (F)× 620; (G, H, L, and P)× 625; (J) ×700; (K) ×850; (M) ×440; (N) ×200; (O) ×450.
Figure 2.
 
Mean E-selectin staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 1 ).
Figure 2.
 
Mean E-selectin staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 1 ).
Figure 3.
 
Mean ICAM staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 2 ).
Figure 3.
 
Mean ICAM staining intensity of iris, CB, choroid, and retina after intravitreal injection of S. aureus and NS. Because of the number of tissues stained in both groups, SDs are not presented (see Table 2 ).
Figure 4.
 
In vitro E-selectin expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in E-selectin expression are noted for all concentrations of RTA (10 and 1 μg/ml, P < 0.001; 0.1 μg/ml, P < 0.02). (B) Statistically significant increases in E-selectin expression are noted for all concentrations of PG (P < 0.0005). Data presented are representative samples of at least triplicate trials.
Figure 4.
 
In vitro E-selectin expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in E-selectin expression are noted for all concentrations of RTA (10 and 1 μg/ml, P < 0.001; 0.1 μg/ml, P < 0.02). (B) Statistically significant increases in E-selectin expression are noted for all concentrations of PG (P < 0.0005). Data presented are representative samples of at least triplicate trials.
Figure 5.
 
In vitro ICAM-1 expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in ICAM-1 expression were noted for RTA concentrations of 10 and 1 μg/ml (P < 0.0001) and a nearly significant increase with 0.1 μg/ml (P = 0.058). (B) Statistically significant increases in ICAM-1 expression were noted for PG concentrations of 10, 1, and 0.01 μg/ml (P < 0.0005) and 0.1 μg/ml (P < 0.04). Data presented are representative samples of at least triplicate trials.
Figure 5.
 
In vitro ICAM-1 expression after 4-hour stimulation with S. aureus RTA and PG. (A) Statistically significant increases in ICAM-1 expression were noted for RTA concentrations of 10 and 1 μg/ml (P < 0.0001) and a nearly significant increase with 0.1 μg/ml (P = 0.058). (B) Statistically significant increases in ICAM-1 expression were noted for PG concentrations of 10, 1, and 0.01 μg/ml (P < 0.0005) and 0.1 μg/ml (P < 0.04). Data presented are representative samples of at least triplicate trials.
Table 1.
 
E-selectin Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Table 1.
 
E-selectin Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Noninjected 6 Hours 24 Hours 48 Hours 72 Hours
NS WT NS WT NS WT NS WT
CB 0.05 ± 0.15 2.33 ± 1.0 2.14 ± 0.75 0.25 ± 0.54 1.33 ± 0.99 0.21 ± 0.35 0.70 ± 0.68 0.38 ± 0.32 0.29 ± 0.37
Iris 0.02 ± 0.08 2.58 ± 1.0 2.46 ± 0.63 0.38 ± 0.49 1.73 ± 0.89 0.64 ± 0.64 1.10 ± 0.74 0.28 ± 0.25 0.18 ± 0.31
Choroid 0.05 ± 0.15 0.81 ± 0.65 1.04 ± 0.48 0.25 ± 0.43 1.25 ± 0.88 0.46 ± 0.49 0.65 ± 0.41 0.20 ± 0.35 0.11 ± 0.28
Retinal vessels 0.00 0.42 ± 0.52 1.32 ± 0.64 0.06 ± 0.17 1.28 ± 1.0 0.05 ± 0.15 0.80 ± 0.63 0.05 ± 0.16 0.00
Table 2.
 
ICAM-1 Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Table 2.
 
ICAM-1 Staining Intensity of Ocular Structures from Eyes Injected with WT Staphylococcus aureus and NS
Noninjected 6 Hours 24 Hours 48 Hours 72 Hours
NS WT NS WT NS WT NS WT
CB 0.55 ± 0.27 1.18 ± 0.63 1.31 ± 0.68 1.00 ± 0.47 2.32 ± 0.78 1.08 ± 0.62 2.06 ± 0.73 1.06 ± 0.59 1.36 ± 0.98
Iris 0.77 ± 0.52 1.46 ± 0.65 1.61 ± 0.65 1.20 ± 0.42 2.21 ± 0.94 1.06 ± 0.58 2.19 ± 0.57 1.23 ± 0.61 1.31 ± 0.65
Choroid 0.73 ± 0.45 1.46 ± 0.50 1.06 ± 0.53 0.95 ± 0.46 2.00 ± 0.60 1.17 ± 0.53 1.28 ± 0.86 1.27 ± 0.73 1.06 ± 0.68
Retinal vessels 0.00 0.00 0.06 ± 0.17 0.10 ± 0.32 1.65 ± 0.63 0.23 ± 0.44 0.75 ± 0.65 0.11 ± 0.18 0.13 ± 0.35
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