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(The FASEB Journal. 2007;21:464-474.)
© 2007 FASEB

VLA-4 blockade suppresses endotoxin-induced uveitis: in vivo evidence for functional integrin up-regulation

A. Hafezi-Moghadam1, K. Noda, L. Almulki, E. F. Iliaki, V. Poulaki, K. L. Thomas, T. Nakazawa, T. Hisatomi, J. W. Miller and E. S. Gragoudas

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, Massachusetts, USA

1Correspondence: Angiogenesis Laboratory, 325 Cambridge St., 3rd Floor, Boston, MA 02114, USA. E-mail: ahm{at}meei.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte adhesion to the vascular wall is a critical early step in the pathogenesis of inflammatory diseases and is mediated in part by the leukocyte integrin, VLA-4, which binds to endothelial vascular cell adhesion molecule (VCAM) -1. Here, we investigate VLA-4’s role in endotoxin-induced uveitis (EIU). At various time points (6–48 h) after EIU induction, the severity of the inflammation was evaluated by quantifying cell and protein content in the aqueous fluid, firm leukocyte adhesion in the retinal vessels, and the number of extravasated leukocytes into the vitreous. Functional activation of VLA-4 in vivo was investigated in our previously introduced autoperfused micro flow chamber assay. Firm adhesion of EIU leukocytes to immobilized VCAM-1 under physiological blood flow conditions was significantly increased compared with normal controls (P<0.05), suggesting an important role for VLA-4 in EIU. VLA-4 blockade in vivo significantly suppressed all uveitis-related inflammatory parameters studied, decreasing the clinical score by 45% (P<0.01), protein content in the aqueous fluid by 21% (P<0.01), retinal leukostasis by 68% (P<0.01), and leukocyte accumulation in the vitreous by 75% (P<0.01). Our data provide novel evidence for functional up-regulation of VLA-4 during EIU and suggest VLA-4 blockade as a promising therapeutic strategy for treatment of acute inflammatory eye diseases.—Hafezi-Moghadam, A., Noda, K., Almulki, L., Iliaki, E. F., Poulaki, V., Thomas, K. L., Nakazawa, T., Hisatomi, T., Miller, J. W., Gragoudas, E. S. VLA-4 blockade suppresses endotoxin-induced uveitis: in vivo evidence for functional integrin up-regulation.


Key Words: EIU • VCAM-1 • autoperfused micro flow chamber • leukocyte-endothelial interaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UVEITIS IS A COMPLEX INFLAMMATORY EYE DISEASE that can lead to blindness. It can affect any part of the eye and is characterized by the accumulation of leukocytes in ocular tissues (1 , 2) . Uveitis can have a variety of underlying causes. For instance, acute anterior uveitis is often associated with Behçet’s disease, ankylosing spondylitis, Reiter’s syndrome, and human leukocyte antigen (HLA) B27-associated uveitis as well as other systemic inflammatory diseases (3) . Current therapies for uveitis include corticosteroids and chemotherapeutic agents to reduce inflammation (4) . However, the grave side effects of these drugs, such as increased intraocular pressure (5) or cytotoxicity (6) , limit their use (4 , 5) . Therefore, a new therapeutic strategy is urgently needed (7 , 8) .

Inflammatory leukocyte accumulation has long been known to damage the host in a variety of diseases (9 10 11) . The cascade-like leukocyte recruitment to sites of inflammation begins with transient rolling of leukocytes on the vascular endothelium, followed by leukocyte activation, firm adhesion, and transmigration into the interstitial tissue (12 13 14) . A number of specialized adhesion molecules fulfill distinct functions in the various stages of recruitment, and thus offer attractive molecular targets for treatment of inflammatory diseases. To elucidate the molecular changes to leukocyte surface antigens during diseases such as uveitis, we recently developed the autoperfused micro flow chamber for rodents (15) . In this model a translucent micro flow chamber is placed between the carotid artery and the jugular vein of a live rodent (15) . The animal’s heart continuously perfuses the chamber with blood (15) . Single molecules are immobilized on the chamber surface, and their role in leukocyte recruitment parameters, such as rolling and adhesion, are assessed microscopically under physiological flow conditions. Thus, differences in leukocyte adhesion parameters can be attributed to changes of specific leukocyte surface antigens. This offers ideal conditions for functional studies of antigens on native leukocytes in relevant disease models, bypassing the drawbacks and disadvantages associated with isolated cells (15) .

Integrins are adhesion molecules that play crucial roles during leukocyte recruitment (13) . Members of the integrin superfamily, such as very late antigen-4 (VLA-4) on the surface of leukocytes, bind to immunoglobulin (Ig) counter-receptors expressed on the inflamed endothelium and facilitate rolling and firm adhesion (16 , 17) . The endothelial counter-receptor for VLA-4 is vascular adhesion molecule 1 (VCAM-1) (18) . VLA-4 interaction with VCAM-1 plays a critical role in a number of inflammatory diseases (19) . For instance, in experimental allergic encephalomyelitis, an animal model of multiple sclerosis (MS), prevention of leukocyte infiltration by antibodies against VLA-4 ameliorates signs of disease (20 , 21) . Furthermore, an anti-VLA-4 antibody has been in clinical trials for treatment of MS (22) and Crohn’s disease (23) . However, due to the occurrence of a limited number of progressive multifocal leukoencephalopathies in patients receiving the anti-VLA-4 antibody (Tysabri), the FDA recently issued a safety alert and interrupted the use of all anti-VLA-4 agents in human trials (www.fda.gov/cder/drug/infopage/natalizumab).

Despite VLA-4’s established role in prominent inflammatory diseases of the brain, the role of this molecule in uveitis is not well understood. An increased expression of several adhesion molecules, including VCAM-1, has been shown in the iris of uveitis patients (24) , suggesting that VLA-4 interaction with endothelial VCAM-1 may be involved in recruiting leukocytes during uveitis. Furthermore, inhibition of VLA-4 with a peptide inhibitor was recently shown to have an ameliorating effect on clinical signs of experimental autoimmune uveitis (25) . However, the importance of VLA-4 and its functional activation status during uveitis remains to be investigated.

In this study we aim to elucidate the role of VLA-4 in leukocyte recruitment during uveitis, using the established model of endotoxin-induced uveitis (EIU) in rats (26) . In this model, a footpad injection of lipopolysaccharide (LPS) creates a systemic inflammation, one of its main manifestations being in the ocular tissues, resembling the acute phase of human anterior chamber uveitis. Neutrophils are considered the main leukocyte subtype recruited in EIU (27) , and rat neutrophils are known to express VLA-4 (28 , 29) . However, whether VLA-4/VCAM-1 interaction contributes to leukocyte recruitment in EIU is unknown. Gaining new insights into this question may provide an alternate molecular target in the prevention and treatment of uveitis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Endotoxin-induced uveitis
To investigate the inflammatory response in the anterior and posterior uveitis, we quantified signs of uveitis at various time points in the experimental model of EIU. EIU was induced in Lewis rats via footpad injection of 100 µl LPS (2 mg/ml). This creates a systemic inflammation, one of its main manifestations being in the ocular tissues, resembling the acute phase of human anterior chamber uveitis (26) . Control animals received equal volumes of saline. The experiments were conducted at 0, 6, 24, and 48 h after LPS injection. At each time point, several clinical and experimental parameters were assessed.

Reagents
To block VLA-4, the animals received 5 or 10 mg/kg of the monoclonal anti-rat VLA-4 antibody (clone TA-2, mouse IgG1, Seikagaku America, Associates of Cape Cod, Inc., East Falmouth, MA, USA; lot# 307–13-3–8) intraperitoneally (i.p.) at the same time as or 6 h after the footpad injections, as indicated. The control animals received equal amounts of a nonimmune IgG1 (clone 15H6). LPS was purchased in lyophilized form from Sigma (#L6511; St. Louis, MO, USA).

Clinical evaluation
Using a slit lamp, anesthetized animals were examined in a masked fashion and scored according to the severity of the inflammatory response. The number of leukocytes in the anterior chamber (AC) was evaluated as 0 = no cell, 0.5 = 1–5 cells, 1 = 6–15 cells, 2 = 16–25 cells, 3 = 26–60 cells, 4 = >60 cells observed. The intensity of flare was graded on a 0–4 scale.

In vivo imaging of the retina
For clinical evaluation of the posterior uveitis, rat retinas were visualized using funduscopy (Topcon American Corp., Paramus, NJ, USA) and scanning laser ophthalmoscopy (SLO; Heidelberg Retina Angiograph 2; Heidelberg Engineering, Germany).

Aqueous humor collection
The aqueous humor was drawn by anterior chamber puncture using a 30G needle while the animal was under anesthesia. To obtain leukocyte counts, samples were suspended in Acridine Orange and leukocytes were counted under epifluorescence microscopy. The protein concentration was measured using the Micro Bicinchoninic Acid (BCA)TM Protein Assay Reagent Kit (Pierce, Rockford, IL, USA; #23235).

Concanavalin A (ConA) lectin staining of adherent leukocytes
The chest cavity was opened under deep anesthesia and the left ventricle was cannulated to allow perfusion (30) . The right atrium was opened to achieve outflow. Twenty milliliters of PBS were perfused to clear erythrocytes and nonsticking leukocytes, followed by 20 ml of FITC-coupled ConA lectin (20 µg/ml in PBS, pH 7.4, total concentration 5 mg/kg; Vector Laboratories, Burlingame, CA, USA), which stains adherent leukocytes and the vascular endothelium. The animals were then perfused with PBS alone for 4 min to remove excess ConA. The eyes were enucleated, and retinas were dissected and flatmounted in a water-based fluorescent antifading medium (Fluoromount; Southern Biotechnology, Birmingham, AL, USA) and imaged by fluorescence microscopy (Leica, FITC filter). Only whole retinas in which the peripheral collecting vessels of the ora serrata were visible were used for analysis. Leukocytes in arteries, veins, and capillaries of each retinal tissue were counted in a radius of 3 mm around the optic nerve, and the total number of adherent leukocytes per retina was calculated. All analysis was performed in a masked fashion.

Intravitreal leukocyte accumulation
To assess leukocyte extravasations into the vitreal cavity during EIU, we generated histological slides of the whole eyes of treated rats and obtained the number of leukocytes in the vitreous cavity of these sections. To do so, whole eyes were placed in 4% paraformaldehyde for fixation. Gradual infiltration of graded alcohols was followed by xylene and finally liquid paraffin. Paraffin-submerged eyes were then cooled to harden. Six-micron sections were cut on a Leica microtome and placed on slides. Sections were stained with hematoxylin and eosin (H&E) and coverslipped.

Western blot for retinal VCAM-1
Whole retinas were isolated from enucleated eyes of normal or EIU-treated rats and lysed for 30 min in a solution consisting of 20 mM imidazole hydrochloride, 100 mM KCl, 1 mM MgCl, 1 mM EGTA, 1% Triton, 10 mM NaF, 1 mM sodium molybdenate, and 1 mM EDTA, supplemented with a cocktail of proteinase inhibitors (Roche, Basel, Switzerland). The samples were cleared by microcentrifugation (14,000 rpm for 30 min at 4°C) and assessed for protein concentration. Thirty micrograms of protein/sample were electrophoresed in a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and electroblotted onto nitrocellulose membranes. After 1 h incubation in blocking solution (20% IgG-free normal horse serum in PBS), membranes were exposed overnight at 4°C to the anti-VCAM-1 polyclonal antibody (goat IgG, sc-1504, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing in PBS, the secondary peroxidase-labeled antibody was applied at a 1:10,000 dilution for 1 h at room temperature. The proteins were visualized with the enhanced chemiluminescence technique (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The size and density of the bands were measured using NIH Image software (http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Autoperfused micro flow chamber
Microslides (0.04x0.4 mm cross-sectional dimensions and 2 cm length; VitroCom, New Bedford, MA, USA) were coated with recombinant P-selectin, VCAM-1, or both (50 µg/ml, R&D Systems, Minneapolis, MN, USA) at 4°C overnight. Coated chambers were washed (PBS containing 10% FBS) and pretreated with sterile heparin (B&D, Bedford, MA, USA; #366480) to prevent coagulation (15) . PE10 tubing (B&D, ID 0.28 mm, #427401) and elastic connectors (Dow Corning, Midland, MI, USA; ID 0.51 mm #508–002, ID 0.64 mm #508–003) were used to link the various components of the autoperfused micro flow chamber assembly (15) .

To integrate the chamber assembly with the animal’s circulation, rats were anesthetized with a 1:1 mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) at a dose of 50 mg/kg of ketamine and 10 mg/kg of xylazine, kept at 37°C, and microsurgically prepared for cannulation (31) . The carotid artery and the jugular vein were surgically exposed (15) . Afterward, a microvascular clamp (Roboz, Gaithersburg, MD, USA) was placed on the vessels to temporarily prevent blood flow. A small incision was then made in the vessel wall; the end of a heparinized saline-filled PE10 polyethylene tubing was inserted into the opening and tied in place with surgical thread. Once all connections were securely in place, the vascular clamps were removed, permitting blood to circulate through the chamber assembly for 5 min. Fluid pressure values before and after the chamber were measured throughout the experiments in order to derive hemodynamic forces. Wall shear stress and shear rate were calculated using our previously developed software, Rectflow® (15) .

Live microscopy
An upright microscope (Leica) with saline immersion objectives (20–100x) was used to visualize blood flow through the chamber. Real-time images were recorded with a CCD color camera (Dage, Stamford, CT, USA) mounted on the microscope and connected to a Macintosh computer equipped with a video capture card (Scion Corp., Frederick, MD, USA).

In vitro leukocyte-endothelial adhesion assay
Leukocytes were isolated from peripheral blood of EIU and normal rats using Neutrophil Isolation Media (Cardinal Associates, Santa Fe, NM, USA), then labeled (32) . Neutrophils were resuspended at 2 x 106 cells/ml in RPMI-5% and incubated for 10 min at 37°C with the fluorescent marker, 2',7'-bis-(2-carboxyethyl)-5 (and 6) carboxyfluorescein, acetoxymethyl ester (Molecular Probes, Eugene, OR, USA) in dimethyl sulfoxide (vehicle). Fluorescent-labeled neutrophils were washed once and incubated in RPMI-5% alone or RPMI-5% with a saturating concentration of mAbs for 10 min at room temperature (32) . Then cells were washed again and used at a concentration of 2 x 106 cells/ml immediately in the adhesion assay as described (33 , 34) . Briefly, rat prostate endothelial cells (RPEC) were grown to confluence on tissue culture-treated, plastic microtiter 96-well plates, stimulated for 24 h with 30 ng/ml recombinant human tumor necrosis factor {alpha} (TNF-{alpha}; Genzyme, Cambridge, MA, USA), and incubated for 15 min with RPMI-5% (32) . Neutrophils (50 ml/well) were incubated with RPEC for 10 min at 37°C. Nonadherent cells were removed and the content of the wells was lysed with 10 mM Tris-HCl, pH 8.4, containing 0.1% SDS. Fluorescence was determined in a microtiter plate fluorometer (excitation 485 nm, emission 530 nm) and adhesion was reported as the number of adherent neutrophils/mm2 based on a standard curve.

Statistics
Statistical differences between experimental groups were analyzed on the Microsoft Excel software program using Student’s t test. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Time course of anterior eye inflammation during EIU
The time course of anterior uveitis was investigated by cumulative clinical score, which was based on cells in the anterior chamber, flare, and presence/absence of fibrin. The score of the anterior uveitis was at a statistically significant peak 24 h post-LPS injection compared with the untreated animals (0 h: n=4, 0±0; 24 h: n=6, 6.7±0.3; P24h=5.8x10–6) or those evaluated at 6 or 48 h (6 h: n=7, 1±0; 48 h: n=6, 2.7±0.3; P=1.3x10–5 and P=8.7x10–5, respectively). In line with the cumulative clinical score, leukocyte counts and protein content in the aqueous humor of EIU animals were highest at 24 h post-LPS treatment (cells/ml aqueous humor: 0 h: n=6, 1±0.5; 6 h: n=7, 4.3±1.9; 24 h: n=5, 1926±178; 48 h: n=6, 1622±162, protein concentration (mg/ml) 0 h: n=7, 1.6±0.15; 6 h: n=7, 3.3±0.3; 24 h: n=13, 18.8±1.3; 48 h: n=6, 6.7±0.4). However, though the protein concentration was markedly reduced by 48 h (P=0.5x10–6), the number of extravasated leukocytes in the anterior chamber remained at high levels and did not significantly decrease when compared with the values at 24 h (P=0.24).

Dose-dependent inflammatory changes in retinas of EIU rats
Fundus images obtained by SLO or a Topcon Fundus camera 24 h after LPS treatment (200 µg/animal) showed clinical signs of inflammation, such as dilated retinal vessels and haziness, when compared with the untreated condition (Fig. 1 A). Higher LPS doses (400 µg/animal) elicited qualitatively similar, albeit more pronounced, signs of inflammation (data not shown). However, due to increased mortality of animals at the higher dose, the subsequent experiments of this study were performed with 200 µg LPS per animal.


Figure 1
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  		Figure 1. Characterization of the inflammation in the posterior eye during EIU. A) Visualization of retinal characteristics in EIU rats. Fundus images from rats before and 24 h after LPS treatment at 200 µg per animal, using SLO or a Topcon Fundus camera, show characteristic signs of inflammation, such as vasodilation and haziness. B) Time course of retinal leukocyte adhesion during EIU. Results represent average leukocyte accumulation±SEM as obtained from ConA-stained retinal flatmounts. *Statistical significance. C) Visualization of leukostasis in retinal vessels during EIU. Micrographs show representative ConA staining in retinas of EIU animals 24 h post-LPS treatment or vehicle-treated controls. Arrows depict firmly adhering leukocytes.

Time course of posterior eye inflammation during EIU
The inflammatory response in the posterior chamber was studied by quantifying firm leukocyte adhesion in the retinal vessels of normal and EIU rats using the ConA staining technique (Fig. 1B ). At 6 h post-EIU induction, retinal leukostasis was already drastically increased (232±55 compared with 16±6 cells at 0 h, P=0.0001). This early response surprisingly preceded the relatively minor changes in the overall clinical score at 6 h, as well as the lack of extravasated cells in the anterior chamber at this time point, suggesting that retinal leukostasis is perhaps a more sensitive measure for assessment of the early stages of uveitis.

Congruent with the pattern of inflammatory changes in the anterior chamber, the number of firm adhesions in the posterior chamber peaked at 24 h post-LPS treatment (P=0.0003) and decreased significantly by 48 h (P=0.0004) (Fig. 1B ). Representative flatmounts prepared from the retinas of normal and EIU animals show this large number of firmly adhering leukocytes at 24 h post-EIU induction, whereas few leukocytes are found in the vehicle-treated controls (Fig. 1C ).

Molecular mediators of leukocyte accumulation in EIU
To investigate adhesion molecules underlying the increased leukocyte adhesion to the vascular endothelium in EIU animals, we isolated peripheral blood leukocytes (PBL) from EIU and normal animals and quantified the adhesion of these leukocytes to confluent endothelial monolayers in vitro (Fig. 2 A, B). Significantly more PBLs from EIU animals bound to coated inflamed endothelial cells when compared with the adhesion numbers obtained from untreated animals (41±2 and 6±2, respectively, n=6, P<0.01). Furthermore, incubation of PBLs from EIU rats with the VLA-4-blocking antibody reduced their adhesion by 70% (n=6, P<0.001) under static conditions, indicating a role for VLA-4 in leukocyte recruitment during EIU.


Figure 2
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  		Figure 2. Adhesion molecules involved in leukocyte-endothelial interaction during EIU. A) In vitro adhesion of leukocytes to cultured endothelial cells. Average adhesion numbers of isolated rat leukocytes preincubated with anti-VLA-4 mAb or IgG control on LPS- stimulated endothelial cell monolayers. Results are expressed as averages of 6 independent experiments ±SEM. *P < 0.001. B) Representative fluorescence micrographs of the in vitro leukocyte adhesion assay. a) No LPS, control IgG; b) No LPS, +VLA-4-blocking mAb; c) +LPS, control IgG; d) +LPS, antiVLA-4 mAb. C) Western blot of retinal VCAM-1 expression. Each lane represents VCAM-1 expression in retinas of an EIU rat or normal control. D) Densitometry of retinal VCAM-1 Western blots. Bar graphs show average density of the VCAM-1 bands from Western blots of retinal tissues in normal and EIU animals ±SEM. *Statistical significance. E) Leukocyte accumulation in the autoperfused flow chamber. Normal and endotoxin-treated rats were connected to flow chambers with P-selectin and/or VCAM-1 coating at 4 dyns/cm2. Blood flow was maintained for 5 min and the number of firm adhesions on the chamber surfaces was obtained. Bars represent average number of firm adhesions from autoperfused chamber experiments. Coating is indicated by a + or a –. Error bars represent SEM from n = 6 animals per group. F) Visualization of firm leukocyte adhesion in the autoperfused micro flow chamber. Representative micrographs from experiments with P-selectin and VCAM-1 doubly coated chambers. In untreated animals only a few leukocytes interacted with the chamber (normal), whereas in the EIU animals significantly more leukocytes accumulated on the chamber surface (EIU Rat).

Western blots of retinal tissues of normal and EIU animals 24 h post-LPS injection showed significantly higher amounts of VCAM-1 in the EIU animals, as determined by densitometry (n=4 rats in each group, P<0.01) (Fig. 2C, D ), suggesting VCAM-1 may be playing a role in the leukocyte recruitment. However, to investigate whether the elevated endothelial VCAM-1 expression in the EIU animals sufficiently explains the massive leukocyte recruitment to the retinal vessels 24 h after LPS injection or whether changes to leukocyte antigens may be required, we performed experiments in our recently introduced autoperfused micro flow chamber (15) . To ensure the same endothelial-like conditions for both normal and EIU leukocytes, we coated chamber surfaces with the same concentration of VCAM-1, P-selectin, or a mixture of both molecules and integrated each chamber within the circulation of normal and EIU animals (Fig. 2E, F ). This allowed live microscopy of leukocytes originating from the carotid artery of normal or LPS-treated rats passing through the coated chambers and subsequently re-entering the animal’s body through the jugular vein (15) . Firm leukocyte adhesion to immobilized P-selectin was similar in EIU and control animals (43.3±9.7/mm2 and 37.2±15.5/mm2, respectively, P=0.37). However, leukocyte adhesion to VCAM-1 was significantly higher in EIU animals compared with controls (13.1±5.3/mm2 and 2.3±0.8/mm2, respectively, P=0.04). Furthermore, in the P-selectin and VCAM-1 double-coated chambers, firm leukocyte adhesion was dramatically increased in EIU rats compared with normal controls (342.8±44/mm2 and 137±41.3/mm2, respectively, P=0.001) (Fig. 2E, F ). Since VLA-4 is the designated leukocyte counter-receptor for VCAM-1, these results suggest a functional up-regulation of VLA-4 on peripheral blood leukocytes of EIU animals. However, since it is also conceivable that another leukocyte surface antigen besides VLA-4 during EIU may be responsible for the elevated leukocyte accumulation, we used the neutralizing antibody against VLA-4 and quantified the number of firm adhesions of uveitic leukocytes on immobilized VCAM-1 and P-selectin double-coated chambers. Blockade of VLA-4 caused an 80.1% reduction in the adherent leukocytes in the double-coated chambers (n=4, P<0.05, 68.3±4.3/mm2, comparable to the adhesion numbers obtained in the P-selectin-coated chambers). This suggests that the elevated leukocyte accumulation in the double-coated chambers is mainly VLA-4 dependent. Shear forces in all autoperfused micro flow chamber experiments were quantified as described previously (15) and did not differ significantly between the groups (P values ranging between 0.33 and 0.57).

Role of VLA-4 in ocular inflammatory leukocyte recruitment during EIU
To further elucidate the role of VLA-4 in leukocyte recruitment during uveitis, we treated a group of EIU rats with an i.p. injection of a VLA-4-blocking antibody at the same time as the LPS treatment. Anti-VLA-4 treatment significantly reduced the cumulative clinical score obtained in EIU animals 24 h post-LPS treatment (nanti-VLA-4 mAb=10, ncontrol IgG=12, P=0.0001) (Fig. 3 A). The two main components of this score—the clinical flare and the evaluation of the cell occurrence in the slit-lamp examinations—were significantly reduced by 42% (P=0.2x10–3) and 60% (P=0.2x10–5), respectively (Fig. 3B, C ).


Figure 3
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  		Figure 3. VLA-4 blockade significantly reduces clinical signs of EIU. A) Clinical signs of LPS-induced uveitis. Results represent average values of the established cumulative clinical grading scheme for EIU ±SEM. Grading was performed in masked fashion as the sum of three criteria: occurrence of cells in the anterior chamber (0–4), intensity of flare (0–4), and fibrin (0–1). Twelve animals were treated with the control IgG and 10 received the anti-VLA-4 mAb. *P < 0.001. B) Clinical flare score. Results represent average values of the flare grading scheme (0–4) for EIU ±SEM. *P = 0.2 x 10–3. C) Clinical cell score. Results represent average values of the cell grading scheme (0–4) for EIU ±SEM. *P = 0.2 x 10–5.

Furthermore, the number of leukocytes in the anterior chamber of the EIU animals was markedly reduced in the group that received the VLA-4-blocking mAb (nanti-VLA-4 mAb=10, ncontrol IgG=12, P=0.3x10–4) (Fig. 4 A). This was also reflected in the distribution of the animals with a given cell number in their AC (Fig. 4C ). Furthermore, VLA-4 blockade significantly reduced the protein concentration in the anterior chamber of EIU rats (nanti-VLA-4 mAb=10, ncontrol IgG=12, P=0.001) (Fig. 4B ).


Figure 4
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  		Figure 4. VLA-4 blockade significantly reduces inflammatory signs in the AC 24 h after EIU. A) Leukocyte accumulation in the AC. Average leukocyte counts in the AC of EIU rats treated with anti-VLA-4 mAb (n=10) or control (n=12) ±SEM. *P = 0.3 x 10–4. B) Distribution of EIU animals per AC leukocyte counts. Graph depicts number of EIU animals treated with control IgG or anti- VLA-4 mAb that were sequestered into three groups of <500, 500-1000, and >1000 cells/µl fluid obtained from the AC. *P < 0.05. C) Protein concentration in the anterior chamber of EIU rats. Average protein concentration in AC fluid obtained from EIU animals treated anti-VLA-4 mAb (n=10) or control IgG (n=12) ±SEM. *P = 0.001.

To investigate whether VLA-4 also mediates the inflammatory response of the posterior eye, we quantified firm leukocyte adhesion in the retinal vessels of EIU rats treated with a neutralizing anti-VLA-4 mAb or control IgG. Blockade of VLA-4 showed a remarkable 68% reduction in the number of total firm adhesions per retina (nanti-VLA-4 mAb=10, ncontrol IgG=12, P=0.1x10–4) (Fig. 5 A). The retinal veins of the EIU animals accounted for 93% of the total leukocyte adhesions, as depicted in representative micrographs of the ConA-stained retinas (Fig. 5D ). However, the adhesion in both arteries and veins was significantly reduced with VLA-4 blockade (Fig. 5A-D ).


Figure 5
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  		Figure 5. VLA-4-blockade significantly reduces retinal leukostasis 24 h after EIU. A) Retinal leukostasis in EIU rats. Animals were treated with 5 mg/kg of anti-VLA-4 mAb (n=10) or control IgG (n=12) by i.p. injection. *P < 0.001. B) Firm leukocyte adhesion in retinal veins. Treatments and n numbers as indicated in Fig. 5 A. *P = 1.2 x 10–5. C) Firm leukocyte adhesion in retinal arteries. Treatments and n numbers as indicated in Fig. 5 A. *P = 0.004. D) Representative micrographs of retinal leukostasis in EIU rats. Arrows depict firmly adhering leukocytes.

Whereas normal animals show no leukocytes in their vitreous cavity, 24 h post-LPS injection a large number of leukocytes (206±52, n=11) was found in the vitreous of the EIU animals (Fig. 6 A). VLA-4 blockade resulted in a drastic 75% reduction in the number of leukocytes found intravitreally (51±10, P=0.007) (Fig. 6A, B ).


Figure 6
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  		Figure 6. VLA-4-blockade significantly reduces intravitreal leukocyte accumulation 24 h after EIU. A) Intravitreal leukocyte accumulation. Average intravitreal leukocyte numbers obtained from histological sections of the whole eye of EIU rats treated with anti-VLA-4-blocking mAb or control IgG. Paraffin sections (6 µm in thickness) were stained for leukocytes using H&E. n = 11 in either group. *P = 0.007. B) H&E staining of ocular sections from EIU rats. Micrographs depict representative paraffin sections from an animal with EIU (control) and an animal with EIU treated with the VLA-4-blocking antibody. Arrows depict intravitreal leukocytes.

Therapeutic potential of VLA-4 blockade during EIU
To investigate whether VLA-4 blockade post-LPS injection would reverse inflammatory signs of an already initiated uveitis, we injected the VLA-4-blocking mAb or control IgG 6 h after EIU induction. Treating animals with the VLA-4-blocking mAb at 5 mg/kg body wt significantly reduced the number of firm leukocyte adhesions in EIU rats by 85% (nanti-VLA-4 mAb=10, ncontrol IgG=12, P=0.3x10–5) (Fig. 7 ).


Figure 7
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  		Figure 7. VLA-4 blockade reverses inflammatory signs of EIU. 6 h after EIU induction, animals were treated with 5 mg/kg of anti-VLA-4 mAb (n=12) or control IgG (n=10). *P = 6.4 x 10–5. In the higher dose group, 6 h after EIU induction animals were treated with 10 mg/kg of anti-VLA-4 mAb (n=12) or control IgG (n=11). *P = 2.6 x 10–6.

To investigate whether a higher dose of the mAb would further reduce the number of firm adhesions, we injected EIU rats 6 h after LPS treatment with 10 mg/kg of the blocking antibody or a nonimmune control IgG. At this dose, the mAb also reduced the number of retinal firm adhesions by a significant 61.5% (nanti-VLA-4 mAb=12, ncontrol IgG=11, P=0.7x10–4); however, the magnitude of the reduction was significantly less than that in the 5 mg/kg-treated group (nanti-VLA-4(5mg/kg)=10, nanti-VLA-4(10mg/kg)=12, P=0.015) (Fig. 7) .

Treatment of EIU rats with VLA-4-blocking antibody 6 h post-LPS treatment significantly reduced the number of leukocytes in the aqueous humor in both dose groups (p5mg/kg=0.043 and p10mg/kg=0.047). However, the clinical score and protein concentration in the aqueous humor did not differ significantly with either 5 or 10 mg/kg of VLA-4-blocking mAb (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we characterized the inflammatory response during EIU. The anterior and posterior uveitis in rats showed a consistent time course with respect to the measured inflammatory parameters in this model, exhibiting a robust peak 24 h post-LPS treatment. These data suggest that this model is well suited for investigation of acute inflammatory responses in the eye. Among all the measured parameters of the anterior and posterior uveitis, retinal leukostasis was the first to be present and was significantly increased as early as 6 h post-LPS treatment. This suggests that retinal leukostasis may provide a sensitive early criterion for assessment of subclinical uveitis (35) .

In EIU animals, neutrophils are considered to be the main leukocyte subtype recruited to the inflamed ocular tissues during the time points that are relevant to our study, up to 48 h (27) . Since rat neutrophils express VLA-4 (28 , 29) , it is likely that the recruited leukocytes in our experiments are mainly neutrophils, though some may be other leukocyte subtypes known to express VLA-4, namely, eosinophils, monocytes, and lymphocytes (28 , 29) .

The importance of a variety of adhesion molecules to the outcome of EIU has been explored before (36 , 37) . P- and E-selectin (38 , 39) , ß2-integrins (40 , 41) , and ICAM-1 (42 , 43) have been found to be involved in the ocular inflammatory responses to endotoxin. Blockade of P- and E-selectin results in a lower ICAM-1 expression in the eyes of EIU animals, suggesting that selectin blockade may significantly influence the course of intraocular inflammation (39) . Another recently uncovered target for treatment of endotoxin-induced inflammation is lectin-like oxidized LDL receptor-1, an adhesion molecule involved in leukocyte recruitment (44) . These adhesion molecules are also expressed in eyes with posterior uveitis and are probably regulated by cytokines such as TNF-{alpha} (36) . Blockade or lack of each adhesion molecule listed above does not alone abolish leukocyte infiltration to the eye, suggesting that other molecules with a functional overlap participate in this process (37) . Furthermore, the previously reported prominent role of the CD18/ICAM-1 pathway for the leukocyte recruitment in EIU (37 , 40 , 41 , 45) and the surprisingly large impact of VLA-4 blockade on leukocyte recruitment in this model suggests there could be a synergistic effect between the CD18/ICAM-1 path (37 , 40 , 41 , 43) and the VLA-4/VCAM-1 interaction for the adhesion step of leukocyte recruitment during EIU.

Our data show VCAM-1 up-regulation in the retinal tissue of EIU animals. An increased VCAM-1 may explain in part the enhanced recruitment of leukocytes in retinal tissues of EIU animals. However, since the retinal samples include a variety of vascular (i.e., endothelium or pericytes) and nonvascular cells (i.e., retinal neurons and glial cells), the cellular sources of VCAM-1 within the retina cannot with certainty be attributed to the endothelium.

Since it was feasible that, in addition to the VCAM-1 up-regulation, changes on the leukocyte surface may occur during uveitis and contribute to the enhanced recruitment, we investigated potential changes to VCAM-1’s leukocyte counter-receptor, VLA-4. To determine the isolated contribution of leukocyte VLA-4 in EIU animals, we performed micro flow chamber experiments. We were able to keep the immobilized VCAM-1 at a constant level and quantitatively compare the adhesion of normal and uveitic leukocytes under physiological blood flow conditions. Thus, we could attribute the quantitative changes in adhesion to changes in leukocyte counter-receptors during the disease state, a distinction that would not be easily possible in other experimental systems. Indeed, the autoperfused micro flow chamber experiments revealed that significantly more leukocytes from uveitic animals adhered to the immobilized VCAM-1. A similar significant difference between the adhesion of uveitic vs. normal leukocytes to immobilized VCAM-1/P-selectin double coatings was observed, but at dramatically higher levels. This pattern of higher adhesion of uveitic leukocytes to immobilized VCAM-1 alone or in combination with P-selectin indicates that VLA-4 is functionally up-regulated on PBLs of EIU animals.

Furthermore, the increased adhesion of EIU leukocytes on P-selectin and VCAM-1 doubly coated chambers suggests a possible synergistic effect between these molecules during uveitis. These data along with the VLA-4-blocking experiments in the autoperfused micro flow chamber provide novel evidence for a functional up-regulation of VLA-4 during EIU on circulating peripheral blood leukocytes. This conclusion is substantiated by the specific VLA-4-mediated adhesion of EIU leukocytes to activated endothelial monolayers.

With regard to the functional up-regulation of VLA-4, integrins are known to have active ("open") and inactive ("closed") conformational states apart from numerical changes on the leukocyte surfaces (46 , 47) . Either category of changes, numerical or conformational, may result in functional changes, such as changes in firm leukocyte adhesion (46) . Furthermore, other types of changes to integrins, such as differences in the glycosylation-status, are thought to affect integrin function and regulate firm adhesion (48 , 49) . Our experiments support the conclusion that leukocyte changes during EIU occur and have a significant functional consequence, but whether they are due to numerical or conformational VLA-4 changes remains to be investigated.

Our studies further show that VLA-4 blockade significantly ameliorates the inflammatory outcome during uveitis, as evidenced by a significant reduction in the clinical score, the number of cells, and the protein concentration in the aqueous humor, as well as retinal leukostasis and the vitreal leukocyte accumulation. Thus, VLA-4 blockade prior to LPS treatment can profoundly influence the pathological symptoms of uveitis.

In clinical practice, however, therapeutic interventions are usually started after a patient has presented with manifestations of uveitis (35) . Therefore, we also investigated whether application of the VLA-4 mAb during the disease would halt or reverse the progression of the EIU. Indeed, VLA-4 blockade 6 h after the EIU induction significantly reduced leukocyte recruitment to the retinal vessels. Therefore, VLA-4 might be an alternative target for treatment of posterior uveitis or other retinal inflammatory diseases. Indeed, VLA-4 blockade may offer an advantage over the blockade of other adhesion molecules, such as CD18, which failed to reduce the inflammation when applied after EIU induction (41) . Corticosteroids are currently the mainstay of therapy for the acute phase of uveitis; however, they affect many physiological processes and have a wide range of significant ocular side effects, such as cataract, increase of intraocular pressure, and increased susceptibility to microbial infection. Thus, the authors expect that VLA-4 blockade might be applicable as a more specific therapeutic strategy for the acute phase of anterior uveitis than corticosteroids, one that may have fewer or more tolerable side effects. Even though the repeated use of anti-VLA-4 agents in humans may be too risky for treatment of chronic diseases, such as MS or Crohn’s disease, a short-term use of these agents may still be a rational and justifiable strategy for severe cases of uveitis, which otherwise would lead into blindness.

In sum, our findings show a functional up-regulation of VLA-4 during EIU and suggest an important role for this molecule in the pathogenesis of uveitis. Blockade of VLA-4 effectively reduces various inflammatory parameters in vivo and thus may serve as a target for treatment of uveitis.


   ACKNOWLEDGMENTS
 
We would like to thank Deisy Bula, Yianni Paulus, and Nicolas Mitsiades for their help with the experiments. This work was supported by Knights Templar Fellowship grant to E.I. under the mentorship of E. S. Gragoudas and J. W. Miller, NEI core grant EY14104 under the directorship of T. Dryja, and National Institutes of Health grant AI050775 to A.H.M. We are indebted to the Massachusetts Lions Foundation for generous funds provided for laboratory equipment used in this project. We thank Research to Prevent Blindness for unrestricted funds awarded to the Department of Ophthalmology at Harvard Medical School.

Received for publication May 17, 2006. Accepted for publication August 22, 2006.


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 DISCUSSION
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