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Departments of
* Ophthalmology and
Biomedical Physics and Bioengineering, Aberdeen University Medical School, Scotland, United Kingdom
Correspondence: Heping Xu or Isabel J. Crane, Department of Ophthalmology, IMS Building, Aberdeen University Medical School, Foresterhill, Aberdeen AB25 2ZD, U.K. E-mail: h.xu@abdn.ac.uk or i.j.crane{at}abdn.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion molecule • hyaluronic acid • inflammation • experimental autoimmune uveoretinitis • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD44 is an abundant cell-surface glycoprotein expressed on a wide variety of rodent and primate cells, including most hematopoietic, fibroblastoid, neural, and muscle cells [6 ]. Alternative splicing and/or post-translation modifications generate many CD44 isoforms [7 , 8 ]. CD44 isoforms are involved in a wide variety of cellular processes, such as cell-cell and cell-matrix interactions [9 ], cell trafficking [3 ], presentation of cytokines and growth factors to traveling cells [10 ], and the uptake and intracellular degradation of hyaluoronic acid (HA), the principal ligand of this receptor [8 ]. Recently, CD44 has been shown to be an effective proinflammatory therapeutic target in terms of leukocyte homing in several autoimmune diseases, such as collagen-induced arthritis [11 12 13 ], experimental autoimmune encephalomyelitis (EAE) [14 , 15 ], and diabetes [16 ]. It has therefore been suggested that CD44 is involved in the migration of activated cells and their interaction with extracellular matrix during the inflammation [5 ]. In vitro studies have also shown that cells from immunized mice [3 ] or autoimmune disease patients [4 ] can roll in a CD44-HA-dependent manner under physiological shear stress. However, no direct in vivo evidence for CD44-HA-dependent rolling and migration of cells during inflammation has been obtained so far in any of these model systems.
Experimental autoimmune uveoretinitis (EAU) is an organ-specific autoimmune disease of the eye induced by immunization with retinal-specific antigens [17 , 18 ]. Although the precise immunopathogenic mechanisms of EAU are still controversial, the entire process depends on the migration of activated T-lymphocytes and macrophages into the retina and their interaction with retinal matrix. However, the exact mechanism by which activated lymphocytes and macrophages cross the blood-retinal barrier (BRB) remains unclear. Recently, we have developed an in vivo, noninvasive, real-time cell-tracking system [19 , 20 ] with which leukocyte trafficking at the BRB can be precisely analyzed. We have therefore investigated whether CD44 is directly involved in leukocyte-endothelial cell interactions at the BRB in vivo during EAU using this system. The results show that anti-CD44 monoclonal antibody (mAb) IM7, which recognizes constant epitopes near the extracellular proximal domain of CD44, significantly reduced activated leukocyte rolling and transmigration in the retinal vessels of EAU mice. Furthermore, the severity of EAU was suppressed by repeat injection of IM7 mAb. We also found that the anti-CD44 mAb IM7 induced apoptosis of retinal-infiltrated leukocytes in vivo and in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of EAU and anti-CD44 mAb treatment
EAU was induced in B10.RIII mice as described previously [21
, 22
]. Briefly, mice were immunized subcutaneously with 50 µg human interphotoreceptor retinoid-binding protein (IRBP), peptide 161–180 (SGIPYIISYLHPGNTILHVD; purity >85%; Sigma Chemical Co., Cambridge, UK), emulsified with 50 µl Freund’s complete adjuvant (CFA; H37Ra; Difco Laboratories, Detroit, MI) in a total volume of 100 µl. Control mice were immunized with the same volume of phosphate-buffered saline (PBS), instead of IRBP peptide, in CFA.
At day 8 postimmunization (pi), immunized mice were randomly assigned to one of two groups, which received anti-mouse CD44 mAb (IM7; BD Biosciences, Oxford, UK) or an isotype-matched control mAb [rat immunoglobulin G (IgG)2b, A95-1; BD Biosciences). Each mouse received 20 µg anti-mouse CD44 mAb or isotype-control intravenously (i.v.) on days 8–11 pi. Clinical EAU was scored by examining eyes daily from days 9–16 pi using a slit lamp for the anterior segment and a surgical operating microscope for viewing the posterior segment.
Clinical score
A scoring system [23
] was used where 0 = normal; 0.5 = dilated iris blood vessels or few (one to two), very small peripheral focal lesions in the retina; 1 = engorged iris vessels or mild vasculitis in the retina and less than five small focal lesions; 2 = hazy anterior chamber, decreased red reflex, and multiple chorioretinal lesions and/or infiltratins; 3 = moderately opaque anterior chamber but pupil still visible and dull-red reflex; 4 = opaque anterior chamber and obscured pupil, absence of red reflex, proptosis. At scores 3 and 4, the fundus could not be observed through the opaque anterior chamber.
Histopathology
Mouse were killed at day 16 pi, and the eyes were fixed in 2.5% buffered glutaraldehyde (Fisher Chemicals, Leics, UK) and embedded in resin for standard hematoxylin and eosin staining. The intensity of uveoretinitis was evaluated histologically and graded using a modified version of the customized histologic grading system [22
, 24
] by independent observers.
In vivo leukocyte trafficking assay
Leukocyte preparation
A single-cell suspension was prepared from normal, nonimmunized or day 10 pi mouse spleens according to our previous description [20
]. Splenocytes were then resuspended in 20 ml complete medium [Gibco-BRL, Grand Island, NY; RPMI 1640 supplemented with 10% (v/v) fetal calf serum (FCS), 1 mM sodium pyruvate, 4 mM L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin] at 2 x 106 cells/ml. Cells (2x107) in 10 ml were incubated with 40 µg/ml calcein-AM (C-AM; Molecular Probes Europe BV, Leiden, The Netherlands) at 37°C for 30 min. C-AM is nontoxic and has no effect on cell adhesion [25
]. For blocking experiments, cells were incubated with 5 µg/ml IM7 or rat IgG or 100 µg/ml high molecular weight of HA (HMWHA; MW, 6
12x105; Seikagaku Corp., Tokyo, Japan) for 30 min at 37°C before C-AM labeling. Previous studies have shown that this antibody can block the binding of HA to cell-surface CD44 [26
] and down-regulate leukocyte cell-surface CD44 expression [15
, 27
]. Our flow cytometry study also showed that incubation with IM7 for this time induced significant loss of cell-surface CD44, whereas incubation with HMWHA caused the binding of HA to CD44 without changing CD44 expression (see Results).
Scanning laser ophthalmoscopy (SLO)
The technique of SLO in mice has been described in detail elsewhere [19
, 20
]. In brief, day 10 pi mice (i.e., prepeak EAU) were anaesthetized with an intramuscular injection of 0.4 ml/kg Hypnorm (active ingredients, fentanyl citrate and fluanisone; Janssen-Cilag Ltd., Belgium) and 1 ml/kg Diazepam (Phoenix Pharmaceuticals Ltd., Gloucester, UK) intraperitoneally (i.p.). Sodium fluorescein [50 µl 0.05% (v/v); Sigma Chemical Co.] was injected via the tail vein to outline the vessels, followed by 1 x 107 fluorescently labeled cells from normal or day 10 pi mice, untreated, antibody-treated, or HMWHA-treated, in 150 µl complete medium. SLO images were recorded on videotape (S-VHS) for off-line analysis. For each eye, three regions of interest containing one to three veins/venules were recorded for at least 30 min.
Image analysis
Video analysis was performed off-line as described elsewhere [20
]. Rolling (i.e., cells that interacted visibly with the vessel wall and traveled at considerably slower velocity than noncontacting cells in the blood stream) and noninteracting leukocytes were counted in each venule. The rolling efficiency was calculated as the percentage of labeled rolling cells among the total number of labeled leukocytes that entered a venule. The sticking efficiency was determined as the percentage of labeled leukocytes becoming firmly adherent for at least 20 s compared with the total number of labeled leukocytes that rolled in a venule during the same time interval.
Leukocyte infiltration
C-AM-labeled (1x107; 150 µl), antibody-treated, HMWHA-treated, or untreated day 10 pi splenocytes were injected via the tail vein into day 10 pi mice, untreated or pretreated i.p. with 20 U hyaluronidase [16
] (Seikagaku Corp.) 2 h earlier. After cell infusion (20 h), 100 µl 2% (w/v) Evans blue (Sigma Chemical Co.) was injected via the tail vein and allowed to bind for 5–10 min. Evans blue is an acid dye of the diazo group that binds to albumin in the blood. The eyes were then harvested and fixed in 2% (w/v) paraformaldehyde (Agar Scientific Ltd., Cambridge, UK) for 1 h. Retinal whole mounts were prepared according to the method of Chan-Ling [28
]. In brief, the anterior segment of the globe was removed, and the retina was peeled from the choroid. Retinas were washed twice in PBS for 15 min and then spread on clean glass slides and mounted vitreous side-up under coverslips with Vectashield (Vector Laboratory, Burlingame, CA). Samples were observed using a confocal scanning laser imaging system fitted with krypton/argon lasers (Zeiss LSM510; Carl Zeiss, Gottingen, Germany) such that the Evans blue appeared red, and the C-AM stain appeared green.
Flow cytometric analysis of CD44 expression and HA binding
Preparation of leukocytes from mouse spleen and incubation of the splenocytes with HMWHA were performed as described above. After washing with PBS, cells were incubated with 2% bovine serum albumin (BSA)/PBS to block nonspecific binding, followed by biotinylated HA-binding protein (bHABP; Seikagaku Corp.) at room temperature for 30 min. Samples were then double-stained with R-phycoerythrin (PE)-conjugated streptavidin (Caltag Laboratory, South San Francisco, CA) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD44 (IM7, BD PharMingen, Oxford, UK) and analyzed by flow cytometry (Becton Dickinson, San Jose, CA) using CELLQuest software (Becton Dickinson).
Detection of apoptotic cells in retinal-infiltrated leukocyte population
Mice treated with IM7 or rat IgG were killed 18 h after the fourth injection of antibody. Mouse eyes were removed and pooled in ice-cooled RPMI 1640 containing 10% FCS. Retinas were quickly dissected, both retinas from each mouse being pooled as a single sample. Single-cell suspensions were obtained by pressing the retinas through a 100 µm Falcon cell strainer 2360 (Becton Dickinson). To identify the phenotype of apoptotic cells, cells were first stained with FITC-anti-mouse CD11b (PharMingen) and Allophycocyanin anti-mouse CD3 (PharMingen). Apoptosis assay was performed as described previously [29
]. Briefly, cells were resuspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and 105 cells/100 µl were incubated with 5 µl Annexin V-PtdEtn (PharMingen) and 5 µl Via-ProbeTM (PharMingen) for 20 min in the dark at room temperature. Finally, 400 µl binding buffer was added to each tube, and flow cytometric analysis was performed within 1 h of incubation. Ten-thousand events from each sample were captured, and data were analyzed with the CELLQuest program (Becton Dickinson). Apoptotic cells were distinguished from normal and necrotic cells by labeling with Annexin V and exclusion of Via-Probe. Gating was performed in the CD11b and CD3 populations separately.
In vitro apoptosis study
Cells from day 12 pi retinas or spleens were cultured in complete RPMI 1640 in triplicate in a 96-well plate (200 µl/well, 2.5x106/ml). To evaluate the effect of anti-CD44 mAb IM7 on cell apoptosis, cultured cells were treated with 10 µg/ml IM7, rat IgG control, or 100 µg/ml HMWHA. Twenty hours later, cell apoptosis was measured by flow cytometry as described above. Gating was performed in the CD3 population only, as most living macrophages were adherent and not suitable for fluorescein-activated cell sorter analysis.
Analysis of CD44 expressing following IM7 treatment
Lymphocytes from blood, submandibular lymph nodes, and retinas were collected from animals treated in vivo with IM7 or rat IgG control at 12 h after the fourth injection and were stained with PtdEtn-conjugated CD44-specific mAb (clone KM201; Serotec, Oxford, UK). This mAb recognizes an epitope in the N-terminal HA-binding region of CD44, distinct from that recognized by IM7 [12
, 30
]. Splenocytes pretreated with 5 µg/ml IM7 or rat IgG or 1000 µg/ml HMWHA in vitro for 30 min at 37°C were also stained with PtdEtn-conjugated CD44-specific mAb (KM201 or IM7 according to the treatment). In addition, triple-staining for cell-surface CD3, CD11b, and CD44 was performed. Samples were analyzed by flow cytometry (Becton Dickinson).
CD44 and HA-immune localization
CD44 and HA immunohistochemistry was performed on cryosections as described elsewhere [31
, 32
]. In brief, 5 µm eye cryosections were cut, mounted on gelatinized glass slides, and dried thoroughly. The unfixed sections were treated with 6% (w/v) BSA in phosphate buffer (0.1 M, pH 7.2) for 30 min to block nonspecific binding. Sections were incubated with bHABP (Seikagaku Corp.; protein concentration 5 µg/ml), diluted in 6% BSA/Tris-buffered saline (TBS) overnight at 4°C. After washing extensively with TBS, sections were incubated with R-PE-conjugated streptavidin (Caltag Laboratory) and FITC-conjugated anti-mouse CD44 (IM7; BD PharMingen) for 1 h at room temperature. Specificity of bHABP for binding to HA was determined by evaluating the degree of bHABP binding to tissues predigested with hyaluronidase before applying the bHABP probe. Sections were incubated with Streptomyces hyaluronidase (Seikagaku Corp.; 100 turbidity reducing units/ml sodium acetate buffer, pH 6.0) at 37°C for 3 h and were then washed thoroughly with TBS. Tissue sections were analyzed by confocal laser microscopy (Bio-Rad Microsciences MRC 1024, Hemel, Hempstead, UK).
For immunolocalization of CD44 within the vessels, 20 µg FITC-conjugated anti-mouse CD44 (IM7) mAb (BD PharMingen) or rat IgG control (BD PharMingen) in 80 µl PBS was injected via the tail vein and allowed to bind for 10 min, followed by injection of 100 µl 2% Evans blue. The animals were then killed, and retinal whole mounts were prepared as described above. CD44 expression in mesenteric vessels was also examined after the above treatment. The mesenteric tissues were fixed in 2% paraformaldehyde for 30 min and then were mounted as flat mounts on slides for confocal microscopy (Zeiss LSM510) as for retinal tissue.
Statistical analysis
Statistical analysis of EAU scores (clinical scores and infiltration scores) was by Mann-Whitney test. Each mouse (average of both eyes) was treated as one statistical event. The mean numbers of infiltrating cells in the antibody-treated and control groups were compared using Student’s t-test. Differences in rolling and sticking cells between antibody-treated and control groups were compared using the 
2 test. Differences with P< 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–
). In the posterior segment of the eye, HA staining was present in the sclera, on the inner limiting membrane of the retina, the ganglion cell layer, the inner plexiform layer, and intensely in the interphotoreceptor matrix (IPM; Fig. 1C
, ,
), whereas CD44 was positively stained in the outer plexiform layers (OPL) and the junction between outer nuclear layer (ONL) and IPM by mAb IM7 (Fig. 1C
, ,).
|
–). However, in the retina, although the staining of HA was up-regulated and diffuse, it did not always colocalize with CD44 staining (Fig. 1D
, –). All infiltrating leukocytes were intensely stained for CD44. In cases of severe inflammation, areas of high CD44 positivity and HA negativity could be observed (Fig. 1E , –), possibly representing areas of proliferation of infiltrating T cells.
Immunolocalization of CD44 within vessels
Immunolocalization of CD44 within vessels by injection of IM7 via the tail vein showed that normal retinal (Fig. 2A
) and mesenteric vessels have minimal or no staining for CD44 (data of normal mesenteric vessels not shown). During EAU (day 9 pi), most of the leukocytes within the retinal veins and venules were intensively stained for CD44 (Fig. , 2B
2C
and 2E)
. In contrast, only very few CD44-positive leukocytes were found in the retinal arteries, arterioles (Fig. 2B)
, and mesenteric vessels (Fig. 2D)
. In mice with EAU, occasionally, CD44 was positively stained in some spread cells, possibly antigen-presenting cells or endothelial cells, in retinal venules, and in postcapillary venules (Fig. 2C
and 2E)
but not in mesenteric vessels (Fig. 2D) . Some CD44-positive leukocytes were found adhering to the CD44-positive spread cells (Fig. 2E)
.
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To determine whether disrupting the interaction of CD44-HA could affect the final homing of inflammatory cells in the retina, transmigration of labeled cells was observed in retinal whole mounts 20 h after the infusion. The results showed that in vitro treatment with anti-CD44 IM7 or HMWHA significantly reduced the capacity of IRBP-primed leukocytes to transmigrate into the retina (Fig. 4C) . This was further evidenced by administration of HA-specific hyaluronidase. Twenty units of hyaluronidase injected i.p. 2 h before cell infusion also reduced transmigration of retinal leukocytes (Fig. 4C) .
The anti-inflammatory effect of anti-CD44 mAb in EAU
As EAU is characterized by the influx of activated T cells and macrophages into the retina and as CD44-HA interaction is involved in leukocyte trafficking in EAU, we predicted that disturbing this interaction in vivo should suppress signs of the disease overall. The results showed that mice treated daily from day 8 pi shortly before the onset of disease with control rat IgG2b mAb (n=9) developed obvious clinical disease at day 10 pi. In contrast, only two of the anti-CD44 mAb-treated mice (n=6) developed clinical disease at this time point (P<0.05). The clinical score and histological infiltration score for the anti-CD44-treated group remained significantly less than for the rat IgG2b control group during the reminder of the observation period (P<0.05; P<0.01; Fig. 6A
and 6B)
.
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Anti-CD44 IM7 mAb induces the loss of CD44 antigen from leukocytes
Direct analysis of CD44 expression with the anti-CD44 mAb KM201 showed that in vivo treatment with anti-CD44 mAb IM7 induced significant down-regulation of CD44 expression on lymph node cells and blood lymphocytes but only slightly down-regulated CD44 on retinal-infiltrated lymphocytes (Fig. 8A
8B
8C
), confirming other previous observations [15
, 27
].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously shown that human iris, ciliary body, choroids, and retina expressed higher levels of CD44 in acute sympathetic ophthalmitis [33 ]. In the present study, immunohistochemical staining showed that in eyes with EAU, infiltrating leukocytes expressed higher levels of CD44, and this was further confirmed by flow cytometry of retinal-infiltrated CD3+ and CD11b+ cells. In addition, these CD44-positive cells were found specifically sticking to endothelium in retinal venules and postcapillary venules, the sites of cell rolling, sticking, and infiltration in EAU (H. Xu et al., manuscript submitted). Furthermore, we have shown by histochemical staining that the distribution and up-regulation of HA, the principal ligand of CD44, mirror the distinct distribution of CD44, particularly in the anterior segment of the inflamed eye. These findings suggest that the interaction of cell-surface CD44 with HA or other CD44 ligands is critical to the development of destructive inflammation in the eye during EAU.
One of the main functions of CD44 in inflammation is to bind its ligand HA, which anchors the leukocyte on activated endothelial cells and mediates its rolling along the vessel wall. An anti-inflammatory effect of IM7 has been demonstrated in several autoimmune diseases, and it has been suggested that it acts by preventing the entry of inflammatory cells into the tissue [11 , 14 15 16 ]. In the present study, we have shown directly in vivo that CD44 is critical for the rolling of activated cells, as in vitro pretreatment of the cells with anti-CD44 mAb IM7 significantly reduces the rolling efficiency compared with the IgG control antibody, which had no effect. When cells were preincubated in vitro with HMWHA, although cell-surface CD44 was not down-regulated by this treatment, leukocyte rolling efficiency was again reduced, suggesting that the rolling is mediated by the interaction of leukocyte CD44 and HA acting as an anchor to the endothelial cells. This was further supported by the data showing that disruption of CD44-HA interaction with IM7 or HMWHA or hyaluronase significantly reduced the number of cells infiltrating the retina. Our data also excluded the involvement of CD44-HA at the second step (firm adhesion) of the homing cascade.
Previous in vitro studies have demonstrated that HA-mediated leukocyte anchoring to the endothelial cell surface occurs through the binding of CD44 expressed on activated endothelial cells [34 ]. In the present study, retinal vascular endothelial cells did not stain for CD44 even in EAU, although CD44-positive leukocytes could be observed adhering to them. Binding of HA to CD44 should not prevent CD44 detection by IM7 as shown by the flow cytometric study (Fig. 5B) . The fact that CD44 is not detectable on the endothelial cell surface is thus likely to be a result of the sensitivity of this particular technique. Alternatively, it may indicate that there are other mechanisms for HA anchoring to the endothelium in vivo.
To further clarify the involvement of CD44 in leukocyte recruitment in EAU, we treated the animals with anti-CD44 mAb IM7 from day 8, the day that early cell infiltration occurred in EAU (H. Xu et al., manuscript submitted). Our results showed that anti-CD44 mAb IM7 significantly reduced the inflammation in EAU. This finding suggests that the relatively late effector phase (leukocyte-infiltration phase) of the disease is susceptible to CD44 targeting, further supporting the view that CD44 is involved in leukocyte homing at the BRB in EAU.
CD44high expression is associated with a memory phenotype. A previous study in EAE in which lymphocytes within the inflamed central nervous system (CNS) tissue were predominantly memory cells showed that anti-CD44 mAb IM7 treatment could effectively prevent the migration of primed memory lymphocytes into the CNS and suppress the disease, whereas migration to lymph nodes was unaffected [15 ]. It was therefore speculated that CD44 may assume some or all of the function of L-selectin on memory cells after it is lost following transformation from the naïve to the memory phenotype [15 ]. Thus, high expression of CD44 may be important for optimizing lymphocyte attachment or migration across endothelium [12 , 35 ].
However, it is also possible that the reduced inflammation in anti-CD44 mAb IM7-treated mouse retina was a result of increased leukocyte cell death induced by engagement of CD44 by anti-CD44 antibody. Our data support this notion but only for retinal-infiltrated cells and not for splenocytes. In vitro treatment with IM7 but not HMWHA induced significantly more cell death of retinal-infiltrated CD3+ cells as compared with rat IgG treatment, indicating that upon stimulation with IM7, CD44 signal transduction could induce activated leukocyte apoptosis. Previous in vitro studies have shown that anti-CD44 mAb IM7 could induce apoptosis in thymocytes and a murine Th cell line (IP12-7) in the presence of anti-CD3 antibody [36 ], which supports the present observations. In addition, several other studies have shown that CD44 also acts as a costimulatory molecule for T cells, inducing T cell proliferation [36, 37 ] and promoting T cell survival [38 ]. In vivo treatment with IM7 has shown suppressed T cell proliferation [27 ]. Thus, engagement of CD44 by IM7 has a dual effect on preventing infiltration of leukocytes into the retina and also promoting activation-induced cell death, while blockade of CD44 with HA only affects trans-endothelial migration.
We have shown clearly that CD44 is critical for leukocyte recruitment at the BRB during EAU, and this will be an important factor in the anti-inflammatory effect of anti-CD44 treatment by preventing the entry of inflammatory cells into the retina. However, this anti-inflammatory effect will also be mediated by the increased cell death of the retinal-infiltrated leukocytes or possibly by other mechanisms such as cell-matrix interaction, leukocyte movement in the subendothelial space, regulation of leukocyte and parenchymal cell interaction, and proliferation of inflammatory cells. These additional modes of action of CD44 warrant further investigation in this system. Clearly, CD44 may be a valuable therapeutic target in the human form of this sight-threatening disease, endogenous posterior uveitis.
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ACKNOWLEDGEMENTS |
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Received June 24, 2002; revised August 26, 2002; accepted September 20, 2002.
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