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

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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 vas- cular permeability. Expression of intercellular adhesion mole- cule (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 extrav- asation. 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. Investigative Ophthalmology & Visual Science, January 2003, Vol. 44, No. 1 226 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/29/2021 IOVS, January 2003, Vol. 44, No. 1 Leukocyte Trafficking in EAU 227 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, iso- type 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.
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