Breakdown of Blood–Retinal Barrier and Upregulation of Cellular Adhesion Molecules

Leukocyte Trafﬁcking in Experimental Autoimmune Uveitis: Breakdown of Blood–Retinal Barrier and Upregulation of Cellular Adhesion Molecules

Heping Xu, John V. Forrester, Janet Liversidge, and Isabel J. Crane

PURPOSE. To clarify the order of events occurring in the CONCLUSIONS. The sequence of events in EAU appears to be breakdown of the blood–retinal barrier (BRB) in experimental focal adhesion of leukocytes to discrete sites on postcapillary autoimmune uveoretinitis (EAU) and in particular to study the venules, followed by upregulation of adhesion molecules, es- relationships between increased vascular permeability, upregu- pecially ICAM-1 and P-selectin, and breakdown of the BRB, lation of endothelial cell adhesion molecules, and leukocyte leading to transendothelial migration of leukocytes and recruit- adhesion and infiltration during EAU. ment of large numbers of cells to the retinal parenchyma. METHODS. B10.RIII mice were immunized with human inter- These changes occur over a short period of 6 to 9 days pi and photoreceptor retinoid binding protein (IRBP) peptide 161– initiate the process of tissue damage during the following 2 to 180. Changes in the retinal microvasculature were examined 3 weeks. (Invest Ophthalmol Vis Sci. 2003;44:226–234) DOI: on days 3, 6, 7, 8, 9, 10, 16, and 21 postimmunization (pi). 10.1167/iovs.01-1202 Evans blue dye was administered intravenously to assess vascular permeability. Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, P- xperimental autoimmune uveoretinitis (EAU) is a T-cell– selectin, E-selectin, and platelet endothelial cell adhesion Emediated autoimmune disease and serves as an animal molecule (PECAM)-1 was evaluated by in vivo administration of model of human endogenous posterior uveitis (EPU).1 Break- antibody and subsequent immunostaining of retinal whole- down of the blood–retinal barrier (BRB) and infiltration of mounts. Lymphocytes from inguinal lymph nodes of normal inflammatory cells into the retina are fundamental to the de- and chicken ovalbumin (OVA)- or IRBP peptide–immunized velopment of EAU. mice at day 5, 6, 7, 8, and 15 pi were labeled in vitro with The BRB is located at two sites, the retinal pigment epithe- calcein-AM (C-AM) and infused intravenously into syngeneic lium (RPE) and the retinal vascular endothelium, which form recipient mice, which had been immunized with peptide at the the posterior and anterior barrier, respectively. Under normal same corresponding time point. Wholemount preparations of conditions, this barrier restricts the entry of molecules and retinas were observed 24 hours later by confocal microscopy cells into the neuroretina, but during ocular inflammation, to determine the adhesion and infiltration of lymphocytes. lymphocytes cross the BRB and enter the retina in large num- 2,3 RESULTS. The first observation of an increase in vascular perme- bers. At present, whether BRB breakdown is necessary be- ability occurred at day 7 pi and was restricted to focal areas of fore lymphocytes can infiltrate or whether lymphocyte infiltra- the retinal postcapillary venules of the inner vascular plexus. tion results in BRB breakdown during EAU remains unresolved. 4 This progressively extended to the outer vascular plexus at day Lightman and Greenwood suggested that breakdown of the 9 pi. Specific adhesion of leukocytes to the endothelium of BRB in ocular inflammation was a direct consequence of lym- 5 retinal venules of the inner vascular plexus was first observed phocytic infiltration. However, in another study, Luna et al. at day 6 pi. Leukocyte extravasation into the retinal paren- found that breakdown of the BRB occurs before cell infiltra- chyma from these vessels began at day 8 pi and extended to the tion. outer vascular plexus at day 9 pi. The expression of adhesion In general, lymphocyte migration into sites of inflammation molecules increased progressively during the development of depends on the interaction between molecules expressed on EAU. In particular, the adhesion molecules ICAM-1, P-selectin, the surface of the vascular endothelium and the leukocyte. The and E-selectin were expressed predominately in retinal process starts with selectin-mediated rolling of leukocytes on venules, the sites of BRB breakdown, cell adhesion, and extrav- the endothelium, followed by integrin and platelet endothelial asation, from day 7 pi. The increases in expression of ICAM-1 cell adhesion molecule (PECAM)-1–mediated adhesion and and P-selectin were associated both spatially and temporally transendothelial migration.6–9 Matrix metalloproteinases (MMPs) with breakdown of the BRB, cell adhesion, and extravasation. are also involved in transmigration of leukocytes at the site of No increase in expression of P-selectin and ICAM-1 was ob- inflammation.10 Within the retina, the vascular endothelial cell served in either the mesenteric vessels of EAU mice or the is in direct contact with circulating lymphocytes, and interac- retinal vessels of OVA-immunized mice. tions between these cells can directly control leukocyte extravasation. However, little is known about the molecular process of leukocyte recruitment at the BRB during EAU. From the Department of Ophthalmology, Aberdeen University The purpose of this study was to determine the relationship Medical School, Scotland, United Kingdom. between changes in vascular permeability, endothelial cell Supported by Grant 057311 from The Wellcome Trust. expression of cellular adhesion molecules, and leukocyte ad- Submitted for publication December 3, 2001; revised May 9 and hesion and infiltration and the role that these changes play in July 29, 2002; accepted August 9, 2002. the breakdown of the anterior BRB (i.e., the retinal vascula- Commercial relationships policy: N. ture) in EAU. We performed the investigation on wholemount The publication costs of this article were defrayed in part by page preparations of the retina11 and used confocal microscopy, charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. which has the unique advantage of allowing access to and Corresponding author: Heping Xu, Department of Ophthalmol- direct comparison of the different regions of the retinal vascu- ogy, Aberdeen University Medical School, Foresterhill, Aberdeen AB25 lature. BRB breakdown was defined by the leakage of blood 2ZD, Scotland, UK; [email protected]. albumin from vessels, as detected by Evans blue.

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MATERIALS AND METHODS sion molecule [ICAM]-1, isotype control: FITC-conjugated hamster IgG), FITC-conjugated anti-mouse CD62P (P-selectin, isotype control:

Animals FITC-conjugated rat IgG1), purified anti-mouse CD62E (E-Selectin, isotype control: rat IgG ) and FITC-conjugated anti-mouse CD31 (platelet Female B10.RIII mice from the animal facility at the Medical School of 2a endothelial cell adhesion molecule [PECAM]-1, isotype control: FITC- Aberdeen University (8 to 12 weeks old, weight ϳ20 g) were used in conjugated rat IgG ) were purchased from PharMingen (BD Bio- this study. The animals were cared for in accordance with the ARVO 2a sciences, Oxford, UK). The purified anti-mouse CD62E and isotype Statement for the Use of Animals in Ophthalmic and Vision Research control rat IgG were labeled with a fluorescent protein kit (Alexa and under the regulations of the UK Animal License Act 1986 (UK). 2a Fluor 488; Molecular Probes Europe BV). VCAMs were stained in vivo Induction of EAU with the above antibodies, by using a slightly modified version of the method previously described.16,17 Briefly, 40 ␮L (20 ␮g) of each EAU was induced in B10.RIII mice, as described previously.12 Briefly, antibody or isotype control in 80 ␮L PBS was injected through the tail mice were immunized subcutaneously in the inguinal region with 50 vein and allowed to bind for 15 minutes before injection of Evans blue. ␮g IRBP peptide 161–180, (SGIPYIISYLHPGNTILHVD; purity Ͼ85%; The animals were then killed and retinal wholemounts prepared as Sigma, Cambridge, UK) emulsified with 50 ␮L Freund’s complete described earlier. The expression of P-selectin and ICAM-1 in mesen- adjuvant (CFA, H37Ra; Difco Laboratories, Detroit, MI) in a total vol- teric vessels was also observed after the treatment. The mesenteric ume of 100 ␮L. Control mice were immunized with the same volume tissues were fixed in 2% paraformaldehyde for 30 minutes and then of phosphate-buffered saline (PBS), instead of IRBP peptide, in CFA. In were mounted on slides for confocal microscopy. addition, 50 ␮g chicken ovalbumin (OVA; Sigma) was used as the non–retinal antigen-specific immunized control. Confocal Microscopy Identification of Microvascular Changes All retinal wholemounts were examined for Evans blue and either C-AM or FITC by a confocal scanning laser imaging system fitted with To evaluate microvascular permeability in the retina during the devel- krypton-argon lasers (MRC 1024; Bio-Rad Microsciences, Hemel Hemp- opment of EAU, 100 ␮L of 2% (wt/vol) Evans blue dye (Sigma) was stead, UK). Using dual blue and green fluorescence, the Evans blue injected into normal nonimmunized B10.RIII mice (n ϭ 6) and day-5, appeared red and the C-AM or FITC stain appeared green. -7, -8, -9, -12, -16, and -21 postimmunization (pi) mice (n ϭ 5 at each time point) through the tail vein. Evans blue is an acid dye of the diazo Data Analysis group that binds to albumin in the blood, allowing sites of BRB breakdown to be detected readily. Animals were killed by inhalation of The fluorescence intensity of the adhesion molecules was quantified by image-analysis computer software (QWin System; Leica, Wetzlar, Ger- CO2 10 minutes later. The eyes were removed and were immediately immersed in 2% (wt/vol) paraformaldehyde (Agar Scientific Ltd., Cam- many). For each vessel analyzed, the fluorescence intensity in a region bridge, UK) for 1 hour. Retinal wholemounts were prepared according of the parenchyma (without any vessels or artifacts) was measured and to the method of Chan-Ling.13 In brief, the anterior segment of the subtracted from the fluorescence intensities measured inside the lu- globe was removed, and the retina was peeled from the choroid. men of the vessels. Retinal arteries and veins were measured within 1 Retinas were washed twice in PBS for 15 minutes and then spread on mm surrounding the optic disc. Vessels were considered to be inde- clean glass slides and mounted vitreous side up under coverslips with pendent variables. Five to six vessels in each retina were measured, Ϯ antifade medium (Vectashield; Vector Laboratories, Burlingame, CA). and the mean SEM was calculated at each time point. Probabilities when control and EAU mice were compared at each time point were Evaluation of Leukocyte Adhesion and Infiltration calculated with the Dunnett multiple comparison test. P Ͻ 0.05 was considered statistically significant. To evaluate leukocyte adhesion and infiltration during EAU, leukocytes from inguinal lymph nodes of normal nonimmunized B10.RIII mice and mice that had been immunized 5, 6, 7, 8, and 16 days previously RESULTS with peptide were injected through the tail vein into syngeneic normal nonimmunized mice and day-5, -6, -7, -8, and -16 pi mice at the Microscopic Changes in Retinal corresponding time point. Leukocytes from lymph nodes of OVA- Vascular Permeability immunized mice at day 6, 8, and 16 pi were also injected into synge- Vascular Permeability in Control PBS-Immunized neic OVA-immunized mice at corresponding time points after immu- B10.RIII Mice. The BRB was intact in all control PBS-injected nization. Cells were labeled in vitro with calcein-AM (C-AM; Molecular B10.RIII mice. Thus, intravascular injection of Evans blue re- Probes Europe BV, Leiden, The Netherlands) before they were in- sulted in a sharp outline of the retinal vessels, with the dye jected, as described previously.14 In brief, a single-cell suspension was retained within the vessel lumen and no detectable leakage prepared from the draining lymph nodes. Cells were then resuspended into the tissue parenchyma (Fig. 1A). The inner retinal vascular in 20 mL complete medium (RPMI 1640 supplemented with 10% plexus, located in the ganglion cell and nerve fiber layer, [vol/vol] FCS, 1 mM sodium pyruvate, 4 mM L-glutamine, 100 ␮g/mL consists of arteries, arterioles, capillaries, venules, and veins. streptomycin, and 100 IU/mL penicillin; Gibco BRL, Paisley, UK) at 2 ϫ Figure 1G shows the inner retinal plexus in control PBS-in- 106 cells/mL. Cells (2 ϫ 107) in 10 mL were incubated with 40 ␮g/mL jected mice, which was characterized by vessels with a very C-AM at 37°C for 30 minutes. C-AM is nontoxic and has no effect on distinct outline due to retention of Evans blue within the cell adhesion.15 Cells were then washed three times with culture vascular lumen. Figure 1J shows the same region of the retina medium. Cells (1 ϫ 107)in150␮L medium were immediately infused as shown in Figure 1G but focused at the junction of the outer into recipient mice through the tail vein. Twenty-four hours later, the plexiform layer and outer nuclear layer, where the outer retinal leukocyte-infused mice were injected with 2% Evans blue, as described vascular plexus is located. The capillary-sized vessels that make earlier, and the retinal wholemounts were prepared. up this plexus showed excellent retention of dye, with no Immunolocalization of Adhesion Molecules in evidence of fluorescence in the tissue parenchyma and a very distinct outline of the vessel. Retinal Vessels Vascular Permeability in EAU. With the Evans blue dye Fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD106 (vas- technique, the first evidence of breakdown of the BRB with cular cell adhesion molecule [VCAM]-1, isotype control: FITC-conju- leakage of the dye into the retinal parenchyma occurred at day

gated rat IgG2a), FITC-conjugated anti-mouse CD54 (intercellular adhe- 7 pi in animals without clinical evidence of EAU at this stage.

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FIGURE 1. Evans blue-albumin complex leakage in wholemount retinas of control and peptide-immunized mice during the development of EAU. Evans blue was injected through the tail vein 10 minutes before death. (A, G, J) Normal PBS-immunized mouse: retinal vessels were sharply outlined, and there was no Evans blue-albumin complex leakage from the vessels in both inner (G) and outer (J) retinal vascular plexi. (B, H, K) Day-7 pi mouse: Evans blue-albumin complex leakage was seen focally in retinal venules and PCVs (arrows) but was limited to the inner retinal vascular plexus (H). There was no leakage in the outer retinal vascular plexus (K, same ﬁeld as H). (C, D, I, L) Day-9 and -10 pi mice: the number and extent of Evans blue-albumin complex leakage increased as the disease progressed (arrows), both inner (I) and outer (L) retinal vascular plexi were involved. (E, F) Day-16 and -21 pi mice: as disease resolved, leakage of Evans blue was reduced. Scale bar, 100 ␮m.

At day 7 pi, 6 of the 10 retinas contained obvious sites of focal and persisted through day 21 pi in all retinas (Figs.1C–F) and dye leakage in venules and postcapillary venules (PCVs; was mainly located at the retinal veins, venules, and PCVs. Both Figs.1B, 1H) of the inner retinal vascular plexus, although the the outer and inner retinal vascular plexi were involved at this frequency of these sites was limited. In contrast to the inner stage with leakage of Evans blue from the vessel lumen result- retinal plexus, the outer retinal plexus showed no detectable ing in a pronounced blurring of the vessel outline (Figs. 1I, 1L). changes in vascular permeability (Fig. 1K) at this time. How- No leakage was observed in the retinal arteries and arterioles ever, by day 9 pi a marked breakdown of the BRB was evident throughout the observation period. However, these vessels lost

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FIGURE 2. Cell infiltration in the retinal parenchyma. C-AM–labeled cells were injected into immunized mice through the tail vein 24 hours before death, and Evans blue was injected 10 to 15 minutes before death. Reti- nal wholemounts were observed by confocal laser microscopy. (A) Con- trol PBS-immunized mouse showing C-AM–labeled cells sticking in the capillaries of the outer vascular plexus. No cell infiltration was detected. (B) Day-6 pi retina showing C-AM–labeled cells sticking along the venules of the inner vascular plexus with no cell infiltration. (C, D) Day-8 pi retina showing many cells sticking in a retinal vein and two cells infiltrating into retinal parenchyma (C). (D) No cell infiltration was detected in the outer vascular plexus. (E, F) Day-9 pi mouse: cells from the vessels showing BRB breakdown infil- trated both the inner (E) and outer (F) vascular plexi.

their natural curvature and became straighter and narrower retinal veins or venules 1 hour or even 24 hours after cell from days 10 to 21 pi (Figs. 1D–F), suggesting an increase in infusion. vascular tone in these vessels. IRBP Peptide-Immunized Mice. The first evidence for leukocyte adhesion to vascular endothelium of veins and Leukocyte Adhesion and Infiltration during EAU venules occurred at day 6 pi. Although the total number of Nonimmunized B10.RIII Mice. In control nonimmunized detected cells in the retina was not significantly increased B10.RIII mice infused with normal labeled cells, only a few when compared with the normal control nonimmunized recip- leukocytes (11.67 Ϯ 1.80/retina) were detected in whole- ient mice (Table 1), the cells’ preferential location to focal sites mounts within the lumen of retinal vessels 24 hours after cell in retinal veins and venules of the inner vascular plexus was infusion, mostly in the capillaries of the outer retinal plexus clear (Fig. 2B, Table 1, P Ͻ 0.05 and P Ͻ 0.001, respectively). (Fig. 2A, Table 1). Very few cells were detected in the veins or A significant increase in the total number of leukocytes sticking venules and none in the retinal parenchyma. Previous studies within the lumen of retinal vessels was apparent in day-7 pi (Xu et al., manuscript submitted) also showed that infusion of mice (Table 1), but was still restricted to the veins and venules cells from immunized animals (day 12 pi) into normal control of the inner retinal vascular plexus. No cell infiltration of the mice was not associated with significant leukocyte adhesion to retinal parenchyma was detected. At day 8 pi, the number of

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TABLE 1. C-AM Labeled Leukocytes within the Retina of B10.RIII Mice during the Development of EAU, 24 Hours after Cell Injection

Cell Number Location

Time Point Total Inﬁltrating Arteries Veins Venules/PCVs Capillaries

Day 0 11.67 Ϯ 1.80 0.00 Ϯ 0.00 0.00 Ϯ 0.00 1.17 Ϯ 0.48 1.67 Ϯ 0.21 8.83 Ϯ 1.42 Day 6 16.33 Ϯ 2.57 0.00 Ϯ 0.00 0.00 Ϯ 0.00 4.83 Ϯ 1.38* 9.17 Ϯ 1.25‡ 2.17 Ϯ 0.31† (16.00 Ϯ 5.06) (0.00 Ϯ 0.00) (0.17 Ϯ 0.41) (4.17 Ϯ 1.94*) (7.17 Ϯ 2.48*) (4.50 Ϯ 1.87*) Day 7 26.50 Ϯ 1.98* 0.00 Ϯ 0.00 0.00 Ϯ 0.00 6.17 Ϯ 0.95‡ 16.67 Ϯ 1.71‡ 3.67 Ϯ 0.80† Day 8 51.83 Ϯ 4.00† 25.50 Ϯ 3.07 0.50 Ϯ 0.34 15.50 Ϯ 1.48‡ 34.00 Ϯ 2.78‡ 3.50 Ϯ 0.62† (15.33 Ϯ 5.57) (0.00 Ϯ 0.00) (0.33 Ϯ 0.52) (3.67 Ϯ 1.21*) (6.50 Ϯ 2.17*) (4.83 Ϯ 2.56*) Day 9 345.70 Ϯ 14.83‡ 301.30 Ϯ 18.40‡———— Day 16 401.30 Ϯ 21.54‡ 357.00 Ϯ 21.64‡———— (13.33 Ϯ 4.13) (0.00 Ϯ 0.00) (0.67 Ϯ 0.82) (2.67 Ϯ 1.37) (3.83 Ϯ 2.23) (6.00 Ϯ 1.67)

Data are expressed as mean cells per retina Ϯ SEM; n ϭ 6. The location of inﬁltrated cells in day 9 and 16 pi mice could not be determined because of their massive diffuse distribution. The difference between control (day 0) and peptide-immunized mice at each point was compared using the Dunnett multiple comparison test. Data from OVA-immunized mice appear in parentheses. * P Ͻ 0.05. † P Ͻ 0.01. ‡ P Ͻ 0.001.

intravascular sticking leukocytes was further increased (Table at day 9 pi (Figs. 3H2, 4), with a lesser increase on arteries and 1). A small number of such cells had undergone extravasation, arterioles but none on capillaries (data not shown). The outer and the cells were apparent near the vessels of the retinal retinal vascular plexus, however, displayed only weak upregu- parenchyma (Table 1, Fig. 2C). However, this change was lation of ICAM-1 expression between days 9 and 16 pi (data not limited to the veins and venules of the inner retinal vascular shown). The expression of ICAM-1 was detected on the endo- plexus where the BRB was breached, as shown by concomitant thelial luminal surface (Fig. 3H3). In ICAM-1–positive veins and focal leakage of Evans blue dye (Figs. 2C, 2D). The number of venules, blood cell aggregations were observed between days retinal parenchymal infiltrating cells increased dramatically 7 and 21 pi, and ICAM-1 also stained positively on the surface from day 9 pi onward (Table 1). Both the inner and outer of aggregations (Fig. 3P). retinal vascular plexi were involved at this time point (Figs. VCAM-1 Expression. In control mice, VCAM-1 was ex- 2E, 2F). pressed more highly on retinal arteries and arterioles than on OVA-Immunized Mice. Preferential location of injected retinal veins and venules of the inner vascular plexus (Fig. leukocytes in retinal veins and venules of the inner vascular 3D2). Between days 9 and 16 pi, there was a progressive plexus was also observed in day-6 pi OVA-immunized mice increase in the expression of VCAM-1 on retinal arteries, veins, (Table 1). However, there was no further accumulation of cells and venules, with less increase in arterioles (Figs. 3I2, 4). The in these vessels even at days 8 and 16 pi (Table 1). No cellular expression of VCAM-1 was also located on the endothelial infiltration was observed in OVA-immunized mice. luminal surface (Fig. 3I3). PECAM-1 Expression. The expression of PECAM-1 in the Expression of Cellular Adhesion Molecules in retinal vasculature of normal mice was similar to that of Retinal Vessels during EAU VCAM-1 and predominated on retinal arteries and arterioles Selectin Expression. P-selectin expression was negligible (Fig. 3E2). Between days 9 and 16 pi, there was a slight in the retinal vessels of normal (Fig. 3A2) and day-3 pi mice increase in the expression of PECAM-1 on retinal arteries, (Fig. 4). From day 7 pi onward, the expression of P-selectin on veins, and venules and on capillaries of the inner vascular veins and venules of the inner retinal vascular plexus increased plexus (Figs. 3J2, 4). No obvious expression of PECAM-1 was progressively (Figs. 3F2, 4), with only a weak increase in detected on the outer vascular plexus (Fig. 3O2). The expres- expression on arteries and arterioles and none on capillaries sion of PECAM-1 was specifically localized to the intercellular (data not shown). However, P-selectin was not upregulated in junctions of endothelial cells (Fig. 3J3). the outer vasculature during the course of the disease (data not None of the isotype control antibodies resulted in staining shown). The staining pattern of P-selectin appeared as small of the retinal vasculature or parenchyma. granules located on the endothelium (Fig. 3F3). Expression of Adhesion Molecules in Mesenteric E-Selectin Expression. Similar to P-selectin, E-selectin Vessels in EAU staining was negligible in all retinal vessels of normal (Fig. 3B2) and day 3 pi B10.RIII mice (Fig. 4). Between days 7 and 16 pi, To determine whether the upregulation of the studied adhe- there was a progressive increase in the expression of E-selectin sion molecules in EAU is retinal vessel specific, the expression on retinal veins and venules, peaking at day 9 pi (Figs. 3G2, 4). of P-selectin and ICAM-1 in the mesenteric vessels of normal Expression of E-selectin was also located on the endothelial and day-9 pi B10.RIII mice was studied. P-selectin was not surface and appeared granular (Fig. 3G3). No expression of detected in the mesenteric vessels of normal (Fig. 3K2) or E-selectin was detected in the retinal arteries, arterioles, and day-9 pi EAU mice (Fig. 3L2). ICAM-1 was positively stained in capillaries, even at the later stage of the disease from days 9 to the mesenteric vessels of normal mice (Fig. 3M2). There was 16 pi (data not shown). no significant enhancement of ICAM-1 (Fig. 3N2) expression in ICAM-1 Expression. In control, nonimmunized B10.RIII the mesenteric vessels of day-9 pi mice. mice, ICAM-1 was positively identified in the veins and venules of inner retinal vessels but stained weakly in arteries and Expression of Adhesion Molecules in Retinal arterioles and was negative in capillaries (Fig. 3C2). From days Vessels of OVA-Immunized Mice 7 to 16 pi, the expression of ICAM-1 increased progressively on To investigate whether the upregulation of adhesion molecules veins and venules of the inner retinal plexus (Fig. 4), peaking is EAU specific, the expression of ICAM-1 and P-selectin in

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FIGURE 3. Expression of adhesion molecules in the retinal and mesenteric vessels of normal nonimmunized and peptide-immunized mice. FITC-conjugated antibody and Evans blue dye were injected through the tail vein. Retinal wholemounts were prepared for confocal observation. Red, Evans blue showing vessels (1); green, FITC-conjugated antibody showing adhesion molecule expression (2). Expression in retinal vessels of normal nonimmunized B10.RIII of (A1, A2) P-selectin, (B1, B2) E-selectin, (C1, C2) ICAM-1, (D1, D2) VCAM-1, and (E1, E2) PECAM-1. Day 9 pi retina: (F1– F3) upregulation of P-selectin in retinal veins and venules and appearing as small granules on the endothelium; (G1– G3) upregulation of E-selectin in retinal veins and venules and appearing as granules on the endothelium; (H1– H3) upregulation of ICAM-1 on the endothelium of retinal veins and venules; (I1– I3) upregulation of VCAM-1 on the endothelium of retinal arteries; and (J1– J3) upregulation of PECAM-1 on retinal vessels and localization to the intercellular junctions of endothelial cells (J3). Expression in mesenteric vessels of normal nonimmunized mice of (K1, K2) P-selectin and (M1, M2) ICAM-1. Expression in day-9 pi mesenteric vessels of (L1, L2) P-selectin and (N1, N2) ICAM-1. PECAM-1 was expressed speciﬁcally on the inner (O1) but not the outer (O2) vascular plexus (9 days pi). (P) Day-9 pi retina showing blood aggre- gation in an ICAM-1–positive retinal vein. a, artery; v, vein. Scale, 50 ␮m.

retinal vessels was studied in day-9 pi OVA-immunized mice. normal transient arrest (trapping) in the passage of leukocytes Neither ICAM-1 nor P-selectin was significantly enhanced in through the small capillaries is prevented probably by capillary day-9 pi OVA-immunized mouse retinal vessels (Fig. 5). narrowing or occlusion. Adhesion of leukocytes to PCVs was followed 24 hours later by upregulation of adhesion molecules, especially ICAM-1 and P-selectin, and breakdown of the BRB, DISCUSSION leading to transendothelial migration of leukocytes and recruit- Using retinal wholemounts we have been able to covisualize ment of large numbers of cells to the retinal parenchyma. changes in retinal microvascular permeability, the expression These changes occurred over a short period (6–9 days pi) and of endothelial cell adhesion molecules, and the distribution initiated the process of tissue damage by infiltrating lympho- and properties of blood leukocytes. The sequence of events in cytes18 and macrophages19 during the following 2 to 3 weeks. EAU in the B10.RIII mouse appeared to be an initial focal Breakdown of the BRB occurred 24 hours before cell infiltra- adhesion of activated leukocytes to discrete sites on PCVs at 6 tion in this model, in contrast with a previous study that found days pi. Simultaneously, there was a reduction in the number barrier dysfunction occurring concomitantly with lymphocyte of leukocytes observed in small capillaries, suggesting that the infiltration in EAU.4 The differences observed may be due to

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lost their natural curvature and became straight, appearing less compliant during peak disease. However, they were not involved in leukocyte recruitment in EAU. Previously, a similar finding was reported in a soluble-antigen (S-Ag)-induced EAU model in rats, in which breakdown of the BRB and infiltration of inflammatory cells were observed in retinal venules.21 How- ever, the BRB of the inner and outer vascular plexi was not compared in that study. The process that initiates the early increased vascular permeability is unclear. It has been reported that cytokines, such as vascular endothelial growth factor (VEGF),5 tumor necrosis factor (TNF)-␣ and interleukin (IL)-1␤5,22–24 are capable of inducing BRB dysfunction. These factors are also capable of stimulating release of nitric oxide by activated neutrophils and endothelial cells.25 Nitric oxide has been found to contribute in part to the increase in permeability of the BRB.26 The disruption of the BRB in veins, venules, and PCVs of the inner retinal vascular plexus is likely therefore to be the result of the release of such inflammatory agents from the accumulated leukocytes, as discussed in the next section.

Leukocyte Adhesion and Infiltration Preferential location of adhering leukocytes to the endothelium of retinal veins and venules was observed at day 6 pi in both IRBP peptide–immunized and OVA-immunized mice, indicating that this is non–antigen-specific adhesion. However, there was no detectable increase in any endothelial cell adhesion molecules examined at this time point, and what initiates this primary vessel-type specific adhesion of leukocytes is not clear. One possibility is that, 6 days after systemic immuniza- tion, the avidity of adhesion molecules in the veins and venules is increased. It is also possible that other adhesion molecules such as CD44 hyaluronan are involved. We have recently found that CD44 hyaluronan plays an important role in leukocyte homing in EAU (Xu et al., manuscript in preparation). At day 7 pi, further accumulation of leukocytes in retinal FIGURE 4. The fluorescence intensity of adhesion molecule staining in retinal veins (A) and venules (B) during the progression of EAU. The veins and venules was observed in IRBP peptide–immunized difference between control and peptide-immunized mice at each point was compared by the Dunnett multiple comparison test. *P Ͻ 0.05; **P Ͻ 0.01; ***P Ͻ 0.001 versus control (n ϭ 6).

the different animal models used. Alternatively, it is more likely that this inconsistency is due to differences in tissue preparation and technical sensitivity. Compared with conventional histologic techniques, confocal microscopy of retinal wholemounts is more suited to the detection of the initial small, patchy breaches in the barrier and the initial steps toward inﬁltration of lymphocytes, because it allows visualization of the entire retinal vasculature.11,20

Development of Increased Retinal Vascular Permeability in EAU In this study of the inner BRB in EAU, we found that BRB breakdown ﬁrst occurred in the inner vascular plexus and was limited to the retinal veins and venules, and in particular to the PCVs at day 7 pi, later extending to the outer vascular plexus at day 9 pi. The sites of initial BRB breakdown in the inner plexus are the same sites at which leukocyte arrest and adhesion, upregulation of adhesion molecules, and cellular extravasation were detected. Leakage from capillaries was not a signiﬁcant feature. Instead, areas of capillary nonperfusion were observed, suggesting capillary closure by intravascular coagulation and/or cell plugging, although the numbers of FIGURE 5. The intensities of adhesion molecule P-selectin and ICAM-1 leukocytes in capillaries was reduced (Table 1). The BRB at the in retinal veins of normal nonimmunized and day 9 pi OVA- or IRBP retinal arteries and arterioles was intact during the progress of peptide–immunized mice. Data are the mean Ϯ SEM, n ϭ 6. **P Ͻ 0.01 EAU, but these vessels were not unaffected by disease. They versus normal nonimmunized mice (Student’s unpaired t-test).

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mice but not in OVA-immunized mice. The accumulation of expression starting earlier and more closely associated with the leukocytes in IRBP peptide–immunized mice coincided with sites of BRB breakdown, cellular adhesion, and extravasation. an increase in vascular permeability and upregulation of adhe- The upregulation of adhesion molecules was not observed in sion molecules. The mechanism by which this is achieved is the mesenteric vessels of EAU mice and in the retinal vessels of not known. OVA-immunized mice, indicating some degree of specificity in It has been shown that a small number of activated T cells upregulation of adhesion molecules in retinal vessels in EAU. enter the retina of normal rats 12 hours after intravenous Localization and upregulation of P-selectin and ICAM-1 in ret- infusion,27 and it is possible that these cells interact with inal veins and venules in EAU may contribute to the specific perivascular antigen-presenting cells (APCs), with recognition adhesion of activated leukocytes in these vessels and subse- of antigen resulting in the manufacture of proinflammatory quently the breakdown of the BRB. ICAM- and leukocyte func- cytokines and chemokines locally and amplification of the tion-associated antigen (LFA)-1 have been shown to play an response.27 In the present study, we have also shown that important role in the pathogenesis of EAU,32 in that ICAM-1 intravenously injected in vivo primed cells extravasate across expression precedes histologic evidence of inflammation. This the BRB into the retina within 24 hours of injection. A report is in agreement with the present study. In addition, in vivo by Hu et al.28 in a rat model showed that systemic infusion of treatment with anti-ICAM-1 and anti-LFA-1 monoclonal antibod- OVA-activated T cells could induce breakdown of the BRB with ies completely prevents the development of the disease32 or extravasation of a limited number of endogenous non–OVA- significantly reduces its severity.32,33 Activated leukocytes cir- specific T cells into the retina 12 hours later. Using the same culating in the retinal vasculature may upregulate ICAM-1 and technique in the mouse, we did not detect extravasation of P-selectin on the endothelium through cytokines.34 injected lymphocytes into the retinas of mice at day 6 pi when In contrast to ICAM-1 and P-selectin, increases in expression the BRB was still intact (see Fig. 2B), but only from day 8 pi of VCAM-1 and PECAM-1 occurred later and was prominent in when there was evidence of initial breakdown of the BRB. This retinal arteries and arterioles, suggesting that they play differ- agrees with the data of Hu et al., suggesting that extravasation ent roles and may be less important than P-selectin and ICAM-1 of cells into the retina after intravenous infusion may be de- for leukocyte infiltration into the retina during EAU. PECAM-1 pendent on BRB breakdown, which is likely to occur during is believed to be required for neutrophil and monocyte trans- the initial 12-hour lag phase after intravenous injection. How migration both in vivo and in vitro.8,9 It is reported to be adhesion of leukocytes might cause breakdown of the BRB is expressed, at higher levels, on the endothelium of all vessel not known, but it is possible that activated leukocytes interact types.35 However, in our study, it was not detected on the either with perivascular APCs, as suggested by Prendergast et endothelium of the outer vascular plexus, even at days 9 and al.,27 or with antigen-bearing endothelial cells directly, produc- 16 pi when leukocyte infiltration was detected around these ing proinflammatory factors locally. In vitro studies have vessels. shown that retinal vascular endothelial cells could express We conclude therefore that a likely course of events in EAU major histocompatibility complex (MHC) class II and present is as follows. As indicated earlier, the initial event in mouse antigen.29 However, whether this is the case in vivo needs EAU appears to be adhesion of activated lymphocytes which, further investigation. by as yet unknown mechanisms, are followed by a series of Locally produced cytokines change the microenvironment amplification steps involving increased expression of adhesion of retinal veins and venules, leading to upregulation of adhe- molecules and BRB breakdown at the PCVs, with eventual sion molecules and increase in vascular permeability. The ac- more widespread increases in expression of adhesion mole- cumulation of activated cells as a result of an increase in cules and BRB breakdown of the larger venules and veins. adhesion molecules leads to high concentrations of cell-re- What initiates lymphocyte adhesion at day 6 pi is not clear, but leased inflammatory mediators locally and, consequently, fur- possible mechanisms include CD40-CD40L and/or other cog- ther localized changes in permeability and expression of adhe- nate interaction between the endothelium and the lympho- sion molecules. cyte. These require further study. Chemokines are thought to be crucial to leukocyte recruitment and cellular extravasation. We have found that monocyte References chemoattractant protein-1 (MCP-1, CCL2), regulated on activation of normal T-cell–expressed and secreted (RANTES, CCL5), 1. Forrester JV, Liversidge J, Dua HS, Dick A, Harper F, McMenamin and macrophage inflammatory protein (MIP)-1␣ (CCL3) are PG. 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