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1 From the Department of Ophthalmology–Immunology and the 2 Department of Morphology, Netherlands Ophthalmic Research Institute, Amsterdam; the 3 Department Ophthalmology, University of Aberdeen, United Kingdom; the 4 Department Anatomy and Human Biology, University of Western Australia, Perth; the 5 Department Ophthalmology, University of Amsterdam, The Netherlands.
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Abstract |
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METHODS. Lewis rats were immunized with MBP emulsified in complete Freund’s adjuvant (CFA) or with CFA alone. Cellular infiltration of the iris was analyzed at various time points by immunohistochemistry of wholemounts, flow cytometry, and immunoelectron microscopy, by using monoclonal antibodies specific for monocytes/macrophages (ED1), T lymphocytes (R73, W3.25, OX8), T-cell activation markers (OX39, OX40), granulocytes (HIS48), major histocompatibility complex (MHC) class II (OX6), and neurofilament (2H3).
RESULTS. MBP-immunized rats showed development of characteristic monophasic EAE, followed, after resolution of paralysis, by mild self-limited AU. Initially, focal infiltrates of round MHC class II+ and ED1+ cells were found in the iris. During the course of AU, the midiris became massively infiltrated with ED1+ monocytes-macrophages, R73+ T cells, granulocytes (HIS48+), and MHC class II+ cells. The influx of T cells consisted of CD4+ and CD8+ cells, of which only a small fraction (<14 and 11%, respectively) expressed activation markers. The infiltrating cells accumulated in proximity to myelinated and nonmyelinated nerve bundles and in the vicinity of blood vessels in the iris. No evidence was found for demyelination or nerve degradation. Neither EAE nor AU developed in CFA-treated control rats.
CONCLUSIONS. These data show that EAE-associated AU is characterized by a transient mixed cellular infiltrate consisting of monocytes-macrophages, granulocytes, and CD4 and CD8 T cells. The preferential accumulation of inflammatory cells in the vicinity of nerve fibers suggests that AU in this model may result from autoreactivity to nerve antigens.
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Introduction |
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The pathogenic mechanisms underlying EAE-associated AU are still unclear. The initiation of a CD4+ T-cell–mediated autoimmune inflammatory response in the eye requires presentation of the target antigen by major histocompatibility complex (MHC) class II+ antigen-presenting cells (APCs). The rat iris contains a rich plexus of sensory, sympathetic, and parasympathetic nerves,13 of which some are myelinated and contain MBP.13 14 These myelinated nerves in the iris could thus provide the target antigen for activated encephalitogenic T cells. Various potential types of APC are present in the iris. The iris contains a rich network of MHC class II+ dendritic cells,15 16 which possess strong antigen-presenting capacity after cytokine-induced maturation in vitro.17 Moreover, a dense network of macrophages is present in the iris stroma,15 16 which may present antigen to previously activated T cells. Together, these findings suggest that AU may be initiated in a direct manner by encephalitogenic T cells. Evidence against this notion, however, was provided by Verhagen et al.,10 who found that in most cases AU starts after remission of paralysis. Moreover, disruption of the blood–brain barrier and blood–ocular barrier does not abolish the lag period between the onset of EAE and AU.10
The purpose of the present study was to characterize the inflammatory cell infiltrate in the iris during the course of EAE by means of immunohistochemistry, flow cytometry, and electron microscopy. In view of the mild uveitis in this model, we used immunohistochemistry of iris wholemounts to determine the phenotype and distribution of inflammatory cells. Previously, we have shown the considerable advantage of this technique over immunohistochemistry of tissue sections in studies on resident immune cells in the iris15 and in studies on ocular changes in the models of endotoxin-induced uveitis (EIU),16 18 experimental autoimmune uveoretinitis (EAU),19 and experimental melanin protein–induced uveitis (EMIU).20 Flow cytometry was performed to further analyze the phenotype and density of inflammatory cells in the anterior uvea. Electron microscopy was used to provide information about the ultrastructure of infiltrating cells and of the site at which they accumulated. Our results show that EAE-associated AU in Lewis rats was characterized by accumulation of various inflammatory cells in proximity with myelinated and nonmyelinated nerve fibers in the iris.
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Methods |
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Purification of MBP and Induction of EAE
MBP was purified from guinea pig brain according to the method of
Brostoff and Mason,21
with a final cation-exchange
chromatography step (CM-Sephadex; Pharmacia Biotech, Uppsala, Sweden),
according to the instructions of the manufacturer. Analysis of purified
MBP by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) revealed a single band with a molecular weight of
approximately 20 kDa.
Rats were injected on the dorsal surface of one hind foot with 25 µg MBP emulsified (1:1) in complete Freund’s adjuvant (CFA), containing 4 mg/ml Mycobacterium tuberculosis (Difco, Detroit, MI). Control rats were injected with 50 µl of saline-CFA emulsion. After immunization, rats were examined for weight loss, paralysis, and ocular changes using a slit lamp. A pilot experiment revealed that MBP-immunization induces EAE and AU with kinetics similar to those described earlier by Verhagen et al.10
Immunohistochemistry of Iris Wholemounts and CNS
Before whole-body perfusion (to remove cells from the vessels)
rats were injected with 500 IU of intravenous heparin. Rats (n
= 3 per group) were killed at several time points after
immunization (days 0, 10, 14, 20, 25, 31, and 40 for single-staining
immunohistochemistry and days 20, 25, and 31 for double-staining
immunohistochemistry) by an overdose of pentobarbital and flushed
through the left ventricle of the heart with 250 ml cold
phosphate-buffered saline (PBS) and 250 ml cold 4% paraformaldehyde.
Iris–ciliary body (ICB) complexes were dissected from the eyes, as
previously described15
and immediately used for
immunohistochemistry. Spinal cords and optic nerves were removed,
immersed in 20% sucrose in PBS overnight at 4°C and frozen in
optimal temperature cutting compound (OCT; Tissue-Tek; Miles, Elkhart,
IN).
Immunohistochemistry of wholemounts was performed as previously
described (McMenamin20
) using the following
primary monoclonal antibodies (Table 1)
: ED1 (anti-CD68; lysosomal antigen present in monocytes,
macrophages, and most dendritic cells), R73 (anti-
ßTCR), OX8
(anti-CD8), HIS48 (recognizing granulocytes), 2H3 (anti-165-kDa
neurofilament), and OX6 (anti-MHC class II). All monoclonal antibodies
were obtained from Serotec (Oxford, UK), except ED1 (kindly provided by
Christine D. Dijkstra) and 2H3 (Developmental Studies Hybridoma
Bank, Iowa City, IA). Immunolabeled cells were visualized by
horseradish peroxidase–conjugated goat anti-mouse immunoglobulins
(Dako, Glostrup, Denmark) and 3-amino-9-ethylcarbazole (AEC) as
substrate (red reaction product). For double-labeling studies,
biotinylated ED1 (kindly provided by Ed Döpp) and R73 (Serotec)
were used in conjunction with streptavidin-conjugated alkaline
phosphatase (Dako) and fast blue as a chromogen (blue reaction
product). Immunohistochemical analysis of spinal cord and optic nerve
sections was performed in a similar manner with monoclonal antibodies
ED1, R73, and OX6.
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Electron Microscopy
Ultrastructural analysis of infiltrating monocytes and T cells in
the iris was performed by immunoelectron microscopy (EM). Rats (n
= 2 per group) were killed on days 24 and 26 after MBP
immunization. Immunohistochemistry of iris wholemounts was performed as
described earlier with monoclonal antibodies ED1 and R73 (Table 1)
and
diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO) as a
substrate. The DAB reaction product was intensified by a
gold-substituted silver peroxidase method.22
Iris
wholemounts were postfixed in 1% osmium tetroxide, 1% potassium
ferricyanide in 0.1 M cacodylate buffer (pH 7.4), dehydrated in
ethanol, and flat embedded in epoxy resin. Ultrathin sections, cut
perpendicular to the iris, were stained with uranyl acetate and lead
citrate and analyzed with a transmission electron microscope (model
201; Philips, Eindhoven, the Netherlands).
Flow Cytometry
Rats (n = 2–7 at each time point) were killed at days
14, 21, 26, 28, 34, and 38 after immunization and perfused with cold
PBS, as described earlier. Subsequently, the ICB complex was
microscopically dissected from enucleated eyes and dissociated in RPMI
1640 containing collagenase D (1 mg/ml; Boehringer–Mannheim, Mannheim,
Germany), DNase I (0.1 mg/ml; Boehringer–Mannheim), and 10% fetal
calf serum at 37°C for 60 minutes. Cells were washed twice with cold
1% bovine serum albumin (BSA), 0.1% NaN3, and
PBS (FACS buffer; Becton Dickinson, Mountain View, CA) before
immunolabeling.
Two- and three-color flow cytometry was performed as previously
described.23
All reagents and incubations were kept at
4°C. Mouse monoclonal antibodies (Table 1)
specific for rat cell
surface antigens were OX39 (anti-CD25-interleukin [IL]-2 receptor),
OX40 (anti-activation marker on CD4+ T cells),
biotinylated OX1 (anti-CD45-leukocyte common antigen), W3/25
(anti-CD4), OX8 (anti-CD8), and phycoerythrin-conjugated R73
(anti-ßTCR) were all obtained from Serotec. Unconjugated
anti-human factor-I IgG1 (OX21; Serotec), biotinylated anti-tri nitro
pherol IgG1 (PharMingen, San Diego, CA), and
phycoerythrin-conjugated mouse IgG1 (Serotec) were used as isotype
controls. Unconjugated antibodies were detected with
fluorescein-isothiocyanate (FITC)-labeled goat anti-mouse
immunoglobulins (Sigma), and biotinylated antibodies were detected with
streptavidin-Cy5 (PharMingen). Flow cytometry was performed (FACScan;
Becton Dickinson, Mountain View, CA), and 10,000 cells were analyzed.
Regions for analysis were set according to leukocyte forward- and
side-scatter characteristics, backgating to the leukocyte gate, or
isolation of the leukocyte population using a third color.
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Results |
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Inflammation of the Anterior Uvea during EAE
EAE-associated AU was examined clinically and in detail by
immunohistochemistry. Slit lamp biomicroscopy revealed AU in
MBP-immunized rats between days 14 and 40, as previously
described.10
All rats (n = 27) killed at day 25
or later showed development of AU. The ocular inflammatory response was
characterized by signs of miosis and by white infiltrates in the iris
stroma, which were most prominent from days 25 through 31 after
immunization.
Immunohistochemistry of wholemounts was performed to further analyze the dynamics, composition, and severity of the cellular infiltration in the iris. In the iris of CFA-injected control rats, a dense regular network of dendriform and pleomorphic ED1+ and OX6+ cells was found (Figs. 1A 1J ), consistent with previous descriptions.15 No evidence of infiltrating cells was found in the iris of these control rats during the entire experiment (until day 40 after immunization). Similar to untreated rats, the iris of CFA-injected rats contained only a few round ED1+ monocytes-macrophages, R73+ T cells, and HIS48+ granulocytes, scattered throughout the tissue (Figs. 1A 1D 1G 1J) .
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Monoclonal antibody R73, specific for the ßTCR, was used to detect
infiltrating T cells. Infiltration of round R73+
cells was observed in the iris from day 20 (in two of three rats)
through to day 31 (in all rats), but returned to normal by day 40 after
immunization. Infiltrating R73+ cells, similar to
ED1+ cells, were found in the midiris, and a few
cells were present in the periphery (Figs. 1E 1F)
. The largest number
of infiltrating R73+ T cells was found on day 31
after immunization.
Infiltrating HIS48+ cells were found from days 20 through 31 after immunization (in six of nine rats), at the time of massive ED1+ and R73+ cell infiltration. The characteristic cellular staining suggested that the HIS48+ cells were granulocytes and not erythroid cells. Similar to ED1- and R73-positive cells, HIS48+ cells were found predominantly in the midiris (Figs. 1H 1I) .
Focal infiltrates of round OX6+ cells were detected at day 10 (in one of three rats) and day 14 (in two of three rats), before the influx of R73+ T cells. Infiltration of OX6+ cells increased dramatically at later time points (Figs. 1K 1L) . At day 20, resident OX6+ cells obtained a more pleomorphic shape. Because of the high density of cells in the infiltrates, it was not possible to determine the shape of these cells at later time points (Fig. 1L) . Many round or pleomorphic cells could be observed inside blood vessels. The distribution of OX6+ cells paralleled the pattern of ED1+ cells–that is, infiltrating cells were found mainly in the midiris and few in the periphery. At day 40, the number of OX6+ cells had declined compared with the number on day 31. At this time, a large number of pleomorphic OX6+ cells was present in the midiris, scattered throughout the tissue, and foci of OX6+ cells were occasionally detected.
Localization of Inflammatory Cells in the Iris
To determine the distribution of infiltrating cells in relation to
the iris nerves, double staining was performed on iris wholemounts with
ED1 or R73 in combination with a monoclonal antibody (2H3) specific to
neurofilaments. In the peripheral ciliary part of the iris, where
nerves mainly have a radial direction, infiltrating
ED1+ and R73+ cells were
found along thick bundles of nerve fibers and occasionally along small
nerve bundles (Figs. 1M
1N)
, but not along single nerve fibers.
Morphologically, infiltrating cells formed perineuronal cuffs. At sites
where thick nerve bundles crossed large vessels, perineuronal sheaths
were interrupted, and perivascular infiltrates were observed (Fig. 1M)
.
In the midiris, small nerve bundles and single nerves fibers formed an
irregular circular plexus. In this area, inflammation was diffuse and
not obviously perineural. A plexus of thin nerve fibers was also
detected in the sphincter region, but inflammatory cells were not
present in this area.
Transmission electron microscopy (TEM) was performed on ED1- or R73-immunostained iris wholemounts to examine the sites of inflammation and the ocular changes at an ultrastructural level. Nonmyelinated and electron-dense myelinated nerves could be distinguished by TEM. Sections were cut out of areas with accumulations of inflammatory cells in the peripheral region of the iris. ED1+ cells displayed typical labeling of multiple lysosomal compartments (Figs. 3A 3B ), characteristic of activated monocytes-macrophages. Most ED1+ cells had indented nuclei, multiple small protrusions, numerous lysosomes, mitochondria and vesicles, and a well-developed Golgi apparatus (Figs. 3A 3B) . Occasionally, ED1+ cells contained secondary lysosomes, containing membranous material (Fig. 3B) .
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Both infiltrating ED1+ and R73+ cells were observed in the stroma of the iris in proximity with myelinated and nonmyelinated nerve bundles (Figs. 3A 3B 3C 3D 3E) and in the vicinity of blood vessels (Figs. 3A 3C) . No evidence was found for nerve degradation or demyelination.
Most vascular endothelial cells had a normal appearance. At the site of intravascular or extravasated cells, however, endothelial cells possessed numerous small protrusions at the luminal surface (Fig. 3C) . Signs of high endothelium-like venules were not found.
Characteristics of Inflammatory T Cells in the Anterior Uvea
The density and phenotype of infiltrating T cells in the iris
during EAE was analyzed in a separate experiment using flow cytometry.
For these experiments, the ICB was isolated and digested with
collagenase to obtain a single-cell preparation. Few leukocytes were
found in the ICB of CFA-treated control rats (Fig. 4A
) or untreated rats (3.2% ± 1.0% of total ICB cells; mean ±
SD). In accordance with the immunohistochemistry results, flow
cytometry revealed that cellular infiltration of the iris began after
remission of the clinical signs of EAE (Fig. 4B)
. At 28 days after
immunization, the highest number of infiltrating
OX1+ (CD45+) leukocytes was
found in the iris (19.9% ± 6.3% of total ICB cells). At day 21 the
cellular infiltrate consisted predominantly of leukocytes not
expressing R73 (ßTCR), whereas at later time points an almost
equal number of R73+ and
R73- leukocytes was found in the inflamed iris.
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ßTCR+
(R73+) cells in the iris was
CD4+ (Table 2)
. At the peak of disease (day 28 after immunization), 7.8% ± 4.5% of
the total number of ICB cells were
CD4+ßTCR+ cells,
whereas 2.5% ± 1.5% were
CD8+ßTCR+ cells. Only
a minor proportion of the T cells expressed OX39 (CD25/IL-2 receptor
[IL-2R]) or OX40, both markers for T-cell activation.
Expression of OX39 was detected on approximately 10% to 14% of the
CD4+ßTCR+ cells in the
iris at the peak of disease. In most rats, OX40 was expressed on a
smaller number of
CD4+ßTCR+ cells than
was OX39. Expression of OX39 was detected on a small number of
CD8+ßTCR+ cells in the
iris. At day 28 after immunization, only 2% of the
CD8+ßTCR+ T cells
expressed OX39.
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Discussion |
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Although it is plausible that EAE-associated AU is initiated after antigen presentation on MHC class II+ resident APCs in the iris to trace numbers of activated CD4+ T cells, the nature of the early APC events at the onset of EAE-associated AU is largely unknown. The iris contains extensive networks of MHC class II+ dendritic cells and macrophages. Although iris dendritic cells have strong antigen-presenting capacity for naive T cells after cytokine-induced maturation,17 they may be poor APCs in situ in the eye. Recently, it was found that resident macrophages isolated from the iris were more efficient APCs for activated primed T cells than dendritic cells.28 In the present study, resident dendriform ED1+ cells and OX6+ cells in the iris revealed little alteration in shape before the onset of EAE-associated AU, suggesting little or no activation of these cells. Infiltrating monocytes-macrophages (round ED1+ and OX6+ cells) appear to play an important role in the augmentation of EAE-associated AU. They were observed in the inflammatory foci in the iris before the influx of R73+ T cells, and their number increased dramatically during the course of AU. Macrophages are essential effector cells in EAE,24 EAU,29 and EIU.30 In EAE, specific elimination of macrophages produced both a significant suppression of the clinical signs of disease and a marked inhibition of CNS inflammation.24 In the present model, similar depletion experiments may reveal the role of iris infiltrating macrophages, as APCs or effectors or regulatory cells.
Only a small number of the infiltrating CD4+ T cells in the iris during EAE-associated AU were activated as reflected by CD25 expression (<14%) or OX40 expression (<9%). The latter is slightly lower than the result of Adamus et al.,11 but this may be attributable to the removal of intravascular cells by whole-body perfusion before the isolation of the iris performed in the present study. These findings are in line with a number of studies of EAE. In the CNS of rats with EAE, only a minority of total T cells express CD25 or OX40.25 ,6531 32 33 It has been suggested that OX40+ T cells represent the most recently activated antigen-specific T cells at the site of inflammation.32 33 If this is true, only a few activated antigen-specific T cells are present in the iris during EAE, and most infiltrating cells are nonspecific bystander cells in this model. Although the induction of EAE is dependent on activated antigen-specific T cells,34 35 only trace numbers of these cells seem to be required for disease induction.36 Most of the recruited cells in the CNS lesions, however, were found to be superfluous to disease induction.31
TEM revealed the accumulation of activated infiltrating macrophages-monocytes and T cells around nerve fibers in the iris; however, demyelination or nerve degradation was not evident. This is in line with findings of others who have investigated the pathologic changes in the CNS.37 Endothelial cells with numerous processes were the only ultrastructural alterations observed at the site of inflammation. Evidence of high endothelial-like venules–that is, large irregular endothelial cells with increased cytoplasm, shown by Shikishima et al.,4 could not be confirmed in the present study. These authors also described adherent inflammatory cells on the luminal surface of the vascular endothelial cells and entrapment of cells in small vessels. The discrepancy between these results is most likely caused by the addition of Bordetella pertussis to the emulsion used for immunization by Shikishima et al.4 B. pertussis augments vascular permeability by increasing the sensitivity of endothelial cells to mast cell–derived vasoactive amines,38 and enhances EAE39 and EAU.40
Several findings suggest that EAE-associated AU may result directly from MBP-specific T cells that target MBP in the iris. First, MBP is present in the myelin sheath of thick nerve fibers in rat iris.13 Second, T cells accumulate in proximity to myelinated nerve fibers in the iris (Figs. 1 3) , implying that these cells meet their target antigen at this site. Third, T cells, isolated from the eyes of rats with EAE, proliferate in response to MBP.12 Furthermore, adoptive transfer of MBP-specific T cells into Lewis rats induces acute uveitis that resembles the disease induced by MBP immunization.10 11 These findings, however, do not explain the accumulation of infiltrating T cells in the vicinity of nonmyelinated nerve fibers in the iris.
EAE-associated AU may also be caused by T cells specific for an iris antigen other than MBP. Heat shock protein (HSP) 60 has been detected in the CNS during EAE on infiltrating cells and resident cells, including oligodendrocytes and astrocytes,41 and immunization of Lewis rats with MBP in adjuvant has been found to induce CNS infiltration of HSP60-reactive T cells other than MBP-specific T cells.42 Immunization with HSP60 or HSP60 peptides produces iridocyclitis,43 indicating that HSP60 is also expressed in the anterior uvea. Strikingly, T cells isolated from the iris of rats with EAE-associated AU proliferate in response to HSP60 (C. Verhagen, unpublished data, 1995). Taken together, these findings suggest that AU induced by MBP immunization may also result from T-cell reactivity to HSP60. Further studies are needed to determine the antigen specificity of the uveitogenic T cells in this model.
Alternatively, EAE-associated AU may be initiated by cytokines produced during the inflammatory process in the CNS. Because of the arrangement of the vasculature in the anterior uvea,44 circulating cytokines may elicit breakdown of the blood ocular barrier and trigger the adhesion and extravasation of inflammatory cells. Interestingly, raised levels of monocyte chemoattractant protein 1 mRNA and protein have been found in the iris before the onset of EAE-associated AU.27 This mechanism may explain the high number of nonactivated T cells in the iris and the accumulation of infiltrating cells around nonmyelinated nerves during EAE.
In summary, this study describes the characteristics of cellular inflammation in the iris of Lewis rats immunized with MBP. The results show that infiltrating T cells accumulated in the vicinity of nerve fibers in the iris, which suggests that AU may result from autoreactivity to nerve antigens. Whether a similar response is involved in AU associated with MS has yet to be determined. This animal model may help to elucidate the immunopathogenic mechanisms underlying this form of uveitis.
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Acknowledgements |
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Footnotes |
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Supported by Grant 93-141, Vrienden Multiple Sclerosis Research, The Hague, Netherlands (AFdV).
Submitted for publication February 20, 2000; revised April 8, 2000; accepted April 19, 2000.
Commercial relationships policy: N.
Corresponding author: Alex F. de Vos, Department of Immunology, Erasmus University Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. devos{at}immu.fgg.eur.nl
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