Download Resident and infiltrating immune cells in the uveal tract in the

Resident and Infiltrating Immune Cells in the Uveal Tract
in the Early and Late Stages of Experimental Autoimmune
Uveoretinitis
T. L. Butler and P. G. McMenamin
Purpose. To investigate the dynamics of resident and infiltrating immune cells in the choroid and
iris during the early and late stages of experimental autoimmune uveitis (EAU) in Lewis rats.
Methods. Uveoretinitis was induced by footpad injection of crude retinal extract and complete
Freund's adjuvant with concurrent intraperitoneal injection of Bordetella pertussis. Five experimental (EAU) and five control animals (adjuvant alone) were studied at days 5, 7, 9, 11
(prodromal stage) and 42 (late stage) after immunization. Five normal animals and five
animals injected with B. pertussis alone served as further controls. Immunohistochemical localization of resident macrophages, major histocompatibility complex class II (la)+ dendritic
cells (DC), infiltrating mononuclear cells, and T cells was performed on wholemounts of
isolated choroidal and iris tissue.
Results. Double immunolabeling confirmed the presence of distinct networks of macrophages
(591 ± 52 cells/mm2) and DC (746 ± 38 cells/mm2) in the rat choroid. No marked qualitative
and quantitative changes were observed in the density or morphologic appearance of ED2+
resident tissue macrophages in the choroid and iris before clinical onset of ocular disease.
On day 11, infiltration of ED1+ monocytes had occurred in the iris but not in the choroid;
however, marked infiltration of T cells was evident in both choroid (286 ± 161 cells/mm2)
and iris (196 ± 72 cells/mm2). The total density of Ia+ cells was significantly elevated in the
choroid (1152 ± 192 cells/mm2) at day 11, and small, round Ia+ cells were two to three times
more frequent than normal at both sites. The density of T cells and Ia+ cells remained
significantly elevated in the choroid and iris in the late stages of EAU.
Conclusions. These data suggest resident uveal tract macrophages undergo no significant alteration in density in the early stages of EAU and that the earliest site of mononuclear cellular
infiltrate in EAU occurs in the iris. The increased total density of Ia+ cells in the choroid on
day 11 and the presence of significantly increased numbers of small, round Ia+ cells in the
iris and choroid may represent increased trafficking of DC in the eye during uveoretinitis.
Furthermore, the raised numbers of Ia+ cells, concurrent with the influx of T cells, suggests
Ia+ DC and macrophages may act as local antigen-presenting cells in the induction of uveoretinitis. Invest Ophthalmol Vis Sci. 1996;37:2195-2210.
Hixperimental autoimmune uveitis (EAU) is recognized as a useful model of human posterior uveitis.1'2
The target of the autoimmune response in EAU and
human posterior uveitis is the retinal photoreceptors.
The immunopathologic processes in EAU, namely vasFrom the Department of Anatomy and Human Biology, The University of Western
Australia, Nedlands, Perth, Western Australia.
Supported l/y National Health and Medical Research Council.
Submitted for publication December 1, 1995; reviled March 26, 1996; accepted June
4, 1996.
Profnietary interest category: N.
liefnint mjuests: P. G. McMenamin, Department of Anatomy and Human Biology,
The University of Western Australia, Nedlands, Western Australia, 6907 Australia.
culitis and focal retinochoroidal infiltrates, closely
mimic the signs observed in various forms of human
posterior uveitis (see reviews2"5). Experimental autoimmune uveitis is a CD4 T cell-mediated immune response5'6 that can be induced in susceptible species
and strains by injection of a variety of retinal antigens3'7 in association with appropriate adjuvants8 at
sites distant from the eye. More recently, modified
uveal and retinal pigment epithelial melanin has been
used to induce a predominantly anterior segment disease, known as experimental melanin-protein-induced
uveitis.
Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11
The initiation of primary cell-mediated immune
responses requires presentation of antigen within the
groove of the major histocompatibility complex
(MHC) class II molecules on the membrane of antigen-presenting cells (APC), such as dendritic cells
(DC). This peptide-MHC class II complex interacts
with the T-cell receptor14 and is aided by accessory
cell adhesion and co-stimulatory molecules.1516 Although it is well known that uveitis is T cell-mediated,
the cell(s) within the eye responsible for presentation
of retinal autoantigens and induction of human endogenous uveitis is unknown and has been the subject
of much recent research. Although ocular tissues were
long thought to lack a distinct population of APCs
and represent an "immunologically privileged site,"17
recent immunomorphologic studies have revealed extensive networks of macrophages and MHC class II +
DC in the anterior18"21 and posterior portions22 of the
uveal tract (see review23). Recent studies in our laboratory have shown that purified isolated iris DC function
as potent APCs on exposure to cytokine-mediated maturation signals, such as granulocyte-macrophage colony-stimulating factors.24 Studies of isolated choroidal
DC have shown they have similar functional capacity
to act as APCs.25 The retina, by comparison, appears
to lack MHC class II+ cells almost completely in most
experimental animals,26"28 although there are some
reports that human retinal microglia express MHC
class II.2930 However, the appropriate turnover, functional, and double-immunophenotypic studies have
yet to be performed to support the suggestion that
cells of the DC lineage exist within the neural retina.
Histologic, immunopathologic, ultrastructural,
and depletion studies have highlighted the significance of macrophages as effector cells in target organ
damage in EAU (see review4) and experimental autoimmune (allergic) encephalomyelitis.31 Macrophages
also may play an important role in perpetuating local
secondary immune responses by presenting antigens
to T cells within ocular tissues. Macrophages, however,
function poorly as APCs in primary immune responses
when compared to DC because they lack the appropriate secondary messenger signals or co-stimulatory
molecules.14'32 This general phenomenon has been
confirmed recently for rat iris macrophages.24
Although it is well established that the tissues of
the uveal tract are involved in EAU, it is unclear
whether this involvement is primary or secondary.
There is evidence to support the suggestion that the
uveal tract, not the retina, is the initial site of inflammatory cell infiltration into the eye (see reviews4'33).
Furthermore, the presence of a rich network of MHC
class II (Ia) + DC in the uveal tract and their likely
absence from the neural retina suggests an active role
by these cells in disease induction.
The general aim of this study was to perform a
time course study of immune cells in the uveal tract
during the early and late phases of EAU using tissue
wholemounts. It was hoped this technique would provide novel en face perspectives of changes in resident
immune cells and infiltrating inflammatory cells occurring throughout large areas of the choroid and iris,
not achievable by conventional histologic approaches.
The first aim of this study was to extend our previous
data of the morphology, immunophenotype, density,
and distribution of various resident immune cell types
in the normal rat choroid with improved wholemount
and double immunohistochemical techniques. Second, we aimed to determine the response of these
cells and the dynamics of infiltrating inflammatory
cells in the uveal tract during the prodromal stage of
EAU. Finally, we sought to determine the long-term
effects of the disease process on the networks of immune cells in the rat choroid and iris.
MATERIALS AND METHODS
Animals
Sixty-three female Lewis rats (6 to 8 weeks of age,
specific pathogen free), obtained from the Animal
Resources Centre (Murdoch, Australia), were used in
this study. Throughout the study, all procedures conformed to the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research.
Preparation of Crude Retinal Extract and
Immunization
Bovine crude retinal extract (CRE; 2 mg/ml) was prepared as described in our previous studies.34 Experimental animals received a 100 /xl injection of an emulsion, consisting of equal proportions of CRE and complete Freund's adjuvant (Bacto Adjuvant Complete
H37 Ra; Difco, Detroit, MI), subcutaneously into the
right hind footpad. Animals received a concurrent 100
//I intraperitoneal injection of Bordetella pertussis (109
organisms; C.S.L. Parkeville, Victoria, Australia). The
footpad injection corresponded to a dose of 100 fxg
CRE, which in the current and previous studies has
produced a mild to moderate form of EAU (Fig. 1).
Control animals were treated in an identical manner
except that phosphate-buffered saline (PBS) was substituted for CRE in the inoculum. Five control and
five experimental animals were killed at days 5, 7, 9,
11 (prodromal stage) and 42 after immunization (late
stage). The early time points were chosen carefully
after pilot studies revealed that staining reproducibility and quality were severely compromised and quantitation of individual immunolabeled cells was impractical during the peak of the disease (days, 12 to 28)
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Uveal Tract Immune Cells in Experimental Uveoretinitis
and irides were incubated in EDTA (20 mM) for 30
minutes (37°C). Each choroid was dissected from the
sclera as an intact sheet and was divided by radial
incisions into six pie-shaped pieces.
Immunohistochemistry
Day
FIGURE l. The clinical score of ocular disease in experimental autoimmune uveoretinitis as a function of time. Values
represent means (± SEM), and there were a minimum of
five observations per time point.
because the choroid, ciliary body, and iris were swollen
by massive inflammatory cell infiltrates. Two groups
acted as additional controls to provide baseline data:
normal animals (n = 5) and a group that received
only an intraperitoneal injection of B. pertussis (n
= 5).
Clinical Grading of Eye Disease
All eyes were graded clinically on a scale of 0 to 4 widi
the aid of an operating microscope 35 at days 5, 7, 9,
and 11 after immunization until they were killed. Animals studied long term were graded clinically on these
days and on every alternate day thereafter until day
42. Grade 1 (mild) disease was evident as discernible
hyperemia with occasional cells adhering to die lens
and pupil margin. Grade 2 (mild to moderate) disease
was characterized by iris vessel dilation, flare, and iridocyclitis producing large numbers of inflammatory
cells in the anterior chamber; however, the pupil remained visible. Grade 3 (moderate) disease manifested as dense inflammatory cell infiltrates within the
anterior chamber sufficient to obscure the pupil, hypopyon uveitis, and corneal haze. Grade 4 (acute to
severe) disease was similar to grade 3 disease but included intraocular hemorrhage.
Choroidal and Iris Wholemount Preparation
and Immunohistochemistry
Animals were anesthetized (sodium pentobarbitone,
100 mg/kg) before whole body perfusion through the
left ventricle with cold heparinized PBS followed by
cold 2% paraformaldehyde. Eyes were posdixed in
2% paraformaldehyde after enucleation. Each eye was
divided into anterior and posterior segments, and die
lens and capsule were removed carefully. The irides
were dien separated from the cornea-sclera and divided by radial incisions into six equal portions. The
retina was dissected from the posterior segment of
the globe, and the remaining choroid-sclera complex
Samples of choroid and iris tissue from each eye were
incubated with a panel of primary monoclonal antibodies (mAbs). Negative controls included either PBS
alone or an inappropriate mAb (e.g., anti-human macrophage marker). The specificities of primary mAbs
to rat macrophages, DC, and other leukocytes are
listed in Table 1. Single and double immunohistochemical studies were performed as previously described. 2137 For single immunostaining, portions of
tissue were blocked with PBS-bovine serum albuminTween and then were incubated widi the primary
mAb followed by biotinylated sheep antimouse and
streptavidin-horseradish peroxidase (Amersham Laboratories, Buckinghamshire, UK). Primary antibody
incubations were performed at 4°C overnight. Other
steps were performed at room temperature widi incubation times of 30 to 45 minutes. The horseradish
peroxidase was visualized by 3-amino-9-ethyl carbazole
(0.2 mg/ml in acetate buffer pH 5; plus 0.35 /xl/ml
H 2 O 2 [30% wt/vol]). Very few cells displaying endogenous activity were noted in negative controls.
A double-chromogen-immunostaining procedure
diat relies on biotinylation of one primary mAb
(ED2) 38 was used to investigate die extent of co-expression of leukocyte markers on macrophage and DC
populations in normal choroid wholemounts. Choroidal wholemounts were double stained with die following combinations: OX6-ED2, ED3-ED2, and E D 1 ED2. Specimens were incubated widi a nonbiotinylated primary mAb, such as OX6, followed by direcdy
conjugated secondary antibody (sheep anti-mouseTABLE
l. Anti-Rat Monoclonal Antibodies*
Monoclonal
Specificity
OX-6
MHC class II (la) antigen
Glycosylated lysosomal antigen (putative
CD68) in most rat monocytes,
macrophages, and 90% of DC
Membrane differentiation marker:
recognizes glycoprotein on resident/
mature connective tissue
macrophages and small
subpopulation in lymphoid tissues
Macrophage subset in lymphoid organs:
a 175-185 kDa sialoadhesin-like
glycoprotein
CD5/a/3 TCR, pan T<ell
EDI
ED2
ED3
OX-19/R73
* For full description and individual references, see Dijkstra et
;t
"
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Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11
horseradish peroxidase; Amersham). The tissues were
washed three times in PBS and then incubated with
the biotinylated primary mAb (ED2-biotin) localized
by streptavidin-alkaline phosphatase. The ED2-alkaline phosphatase-labeled cells were visualized with the
substrate naphthol AS-MX (0.12 mg/ml) and Fast
blue BB (0.25 mg/ml) in Tris buffer (pH 8.5). Levamisole (0.25 mg/ml) was added to the substrate to block
endogenous alkaline phosphatase activity. Immunolabeled cells were visualized as a blue reaction product.
Cells labeled with the other primary monoclonal (e.g.,
OX6) were visualized using 3-amino-9-ethyl carbazole
(vide supra), which produces a red product. Double
staining or co-localization of both phenotypic markers
resulted in purple cells or occasionally cells that contained discrete blue- and red-stained regions. All visualization agents and chemicals were purchased from
Sigma (St. Louis, MO). Stained choroid and iris-ciliary body wholemounts were mounted in aqueous medium on glass slides and were coverslipped.
Quantitative Analysis
Three animals from each group were subjected to
quantitative analysis. These were chosen on the basis
of disease onset closest to day 11 in that group and
for clarity and reproducibility of immunoperoxidase
staining. Immunopositive cells within choroid and iris
wholemounts were counted using a calibrated eyepiece graticule and a X40 objective lens. The method
of counting immunostained cells in iris has been described.21 Six counts were performed on each piece of
immunostained choroidal tissue—two random fields
from the posterior choroid, two from the mid-choroid,
and two from the peripheral choroid.
Immunostained cells were counted using the forbidden line method.39 Cells were classified into three
morphologic categories2137 as dendriform, pleomorphic, and round-ovoid. The mean area of iris and
choroid tissue sampled for each mAb was 0.504 mm2.
Data from six counts were averaged to give a mean
cell density for each morphologic type and each mAb
per individual animal. Group means represent the
combined means of the animals within each experimental group. All cell counts were performed on
coded slides by a blinded observer (TB). Cell density
data were analyzed using one-way analysis of variance.
Differences between individual groups were subjected
to Rest for invariate small samples. Significance values
indicated differences between experimental and both
normal and control groups unless otherwise stated.
RESULTS
Clinical Evaluation
The onset of clinical disease, as determined by macroscopic clinical examination of the anterior segment,
usually occurred on day 11 after immunization (Fig.
1). Ocular disease was maximal at day 17; by day 32,
manifestations of active disease were minimal or absent. Control animals did not exhibit ocular disease.
Density, Distribution, and Phenotype of
Resident Tissue Macrophages and Dendritic
Cells in the Normal Rat Choroid
In a previous study,22 we described the morphology,
location, distribution, and density of macrophages
and MHC class II (la) + cells in the normal rat choroid
largely on the basis of appearance in sections with only
limited use of tissue wholemounts. Recent advances
in the fixation, preparation, and immunostaining of
choroidal wholemounts allows us to upgrade the description of normal choroidal macrophages and Ia+
DC and, moreover, has revealed important supplementary data on these cells.
The mAb ED2 is a pan-specific resident tissue macrophage marker.40 Resident macrophages were distributed uniformly within the choroid and exhibited a
predominandy perivascular location (Fig. 2). Large
numbers of ED2+ macrophages were associated with
the long posterior ciliary arteries and vortex veins (Fig.
2B). They were predominandy bipolar-pleomorphic
in shape (Fig. 3A). Although dendriform ED2+ cells
were noted, they did not display the extremely
branched dendritic cell processes or ruffled appearance of Ia+ cells (vide infra). Rounded ED2+ cells
constituted less than 1 % of the population (Table 2).
The mean cell density of 591 ± 52 cells/mm 2 observed
in the current study was higher than previously reported (200 ± 13 cells/mm2).22 The discrepancy most
likely was caused by major improvements in the fixation, wholemount preparation, and immunostaining
protocols. For example, in our previous study, choroid
and sclera were stained as one, whereas we now routinely remove the choroid from the sclera, which
markedly improves antibody penetration and staining.
The mAb EDI recognizes a cytoplasmic antigen
associated with phagolysosomes present in monocytes,
most tissue macrophages, and some DC subpopulations.40"42 Immunopositive cells thus tend to have a
"beaded" appearance. Previous studies have shown
that 100% of ED2+ iris tissue macrophages are ED1 + . 37
In the current study, pleomorphic and dendriform
ED1 + cells were present in equal numbers in the choroid, and rounded cells constituted only 4% of the
total (Table 2). The distribution and predominantly
perivascular location of EDI + cells in the normal choroid was identical to ED2+ macrophages. Their mean
density was estimated as 723 ± 47 cells/mm2 (Table 2).
The mAb ED3 is considered a marker of activated
macrophages and reacts strongly with macrophages in
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Uveal Tract Immune Cells in Experimental Uveoretinitis
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2. Choroidal wholemount from a normal Lewis rat stained with the resident tissue
macrophage marker ED2. (A) Low-power view of the entire choroid. Note the long posterior
ciliary arteries orientated in the horizontal plane and the vortex veins peripherally. (B)
Medium-power view to illustrate the dense network of ED2+ macrophages that appear particularly dense around the long posterior ciliary artery (A). V = vortex vein. Magnifications:
(A) X20; (B) X45.
FIGURE
lymphoid tissues and at sites of inflammation.40 There
have been few reports that resident tissue macrophages in nonlymphoid tissues are immunoreactive to
ED3; however, ED3+ macrophages were found to be
more widely distributed in the normal rat choroid
(Fig. 3C) than was previously described.22 The ED3+
macrophages displayed a similar density (580 ± 26
cells/mm2) and morphology to ED2+ resident tissue
macrophages (Table 2, Fig. 3C).
The OX6 mAb is specific for the rat MHC class II
molecule, constitutively expressed on the surface of
DC and some macrophages.43 In die absence of a panspecific anti-rat DC marker, conventional classification
of DC relies on die demonstration of dendriform or
pleomorphic morphology, constitutive MHC class II
(la) expression, and lack of coexpression of pan-macrophage markers.14 In the current study, choroidal
wholemounts stained with the OX6 mAb revealed an
extensive network of dendriform, pleomorphic, and
bipolar Ia+ cells (Fig. 3D). Many appeared close to die
focal plane of the retinal pigment epithelium. Only a
small proportion (5%) of Ia+ cells exhibited a
rounded morphology in normal eyes. The density of
Ia+ cells (746 ± 37 cells/mm2) in the normal rat choroid was considerably higher than that obtained in a
previous study (373 cells/mm2).22 As stated above, this
probably was caused by improved fixation, wholemount preparation, and immunostaining procedures.
Double immunostaining with the combination of
mAb OX6-ED2 confirmed our earlier preliminary
studies22 that Ia+ DC and ED2+ resident tissue macrophages are distinct populations (Figs. 3E, 3F). Macrophages that express la appear purple in the doublestained preparations (Fig. 3F) and form only a minor
subpopulation. The ED1-ED2 double-stained choroidal wholemounts revealed that the majority of ED2+
resident tissue macrophages also contained ED1+ phagolysosomes (Fig 3G); however, there are ED2~, ED1 +
cells that are most likely DC. This is in accordance
with our previous studies, which revealed diat approximately 35% of EDI+ cells in the iris were Ia+. Furthermore, ED3-ED2 double-stained preparations revealed that most ED2+ tissue macrophages were also
ED3+ (Fig. 3H).
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Uveal Tract Immune Cells in Experimental Uveoretinitis
2201
FIGURE 3. (A) High-power view of normal rat choroidal wholemount showing the network
of resident ED2+ tissue macrophages beneath the retinal pigment epithelium, whose regular
hexagonal cell borders can be identified clearly. (B) Medium-power view of EDl-stained
wholemount. (C) ED3+ macrophages in the choroid. (D) Major histocompatibility complex
class II (Ia)+ dendritic cells in choroidal wholemount. Note the irregular distribution, which
contrasts with the regular perivascular location of macrophages. (E,F). Low- and high-power
views of double-color immunohistochemical-stained normal rat choroidal wholemount. The
mAbs ED2 and anti-la (OX6) have been used to co-localize resident tissue macrophages
(blue) and dendritic cells (red). Note that both networks are distinct, and only a few cells
(arrorvheads) coexpress both markers. (G) High-power view of choroidal cells double stained
with ED2 (blue) and EDI (red). Note that almost all cells are double positive (purple). (H)
Choroid double stained with the mAbs ED2 and ED3. Almost all choroidal macrophages
are double positive (purple). Magnifications: (A) X300; (B) X200; (C) X200; (D) X200; (E)
X80; (F) X310; (G) X310; (H) X200.
Morphology and Density of Macrophages and
increase in T-cell density throughout the choroid on
MHC class II Dendritic Cells in the Choroid
the day of disease onset, day 11, in EAU animals (Fig.
During the Prodromal Stage of Experimental
4C, Table 2). A very small subpopulation of unusual
Autoimmune Uveoretinitis
dendriform T cells was observed in day 11 animals
+
(not illustrated).
The density and regular distribution of ED2 resident
tissue macrophages in the choroid was largely unaffected
Choroidal Immune Cells in the Late Phase of
during the prodromal stage of EAU. There was a marginal
Experimental Autoimmune Uveoretinitis (Day 42)
shift from a dendriform to a pleomorphic morphology
The density of macrophages (ED2, EDI, and ED3+)
evident on days 5, 7, and 9 (Table 2). There was no
+
was not conspicuously altered in day 42 EAU animals.
observed increase in the number of ED1 round cells
The total density of Ia+ cells was significantly elevated
(monocytes) on day 11 as would have been expected if
(1233 ±146 cells/mm2) in long-term animals (Table
the mononuclear cell infiltrate characteristic of EAU had
+
2), primarily because of increases in dendriform and
begun. By contrast, a conspicuous ED1 mononuclear
pleomorphic cells. T-cell density in the choroid was
cell infiltrate began in the iris by day 11 (vide infra). The
significandy elevated in day 42 EAU animals (Table 2).
only detectable difference in EDI staining in the choroid
was a generalized adjuvant effect (not observed in pertussis controls) on days 5, 7, and 9 in which there was a slight Morphology, Distribution, and Density of
Immune Cells in the Iris During the Prodromal
shift from a predominantly dendriform to pleomorphic
Stage of Experimental Autoimmune
morphology (Table 2). The pattern of ED3 staining
Uveoretinitis
changed little during EAU apart from a slightly increased
density in experimental and control animals on days 5,
The morphology, distribution, density, and phenotype
7, and 9 (Table 2), which indicated an adjuvant effect
of resident tissue macrophages and Ia+ DC in the nor+
mal rat iris corresponded with our previous studThe density of the la (OX6) cell network in the
choroid increased marginally at days 5, 7, 9, and 11.
ies.20'2137 Iris macrophages were more weakly stained
The majority of these cells were dendriform or pleowith ED3 than those in the choroid.
morphic (Table 2). The total density of Ia+ cells (1152
The network of ED2+ resident tissue macrophages
±192 cells/mm2) in experimental animals at the day
in the rat iris was largely unaffected during the prodroof clinical onset, day 11 (Fig 4A)—although greater
mal phase of EAU. Infiltrates of round ED1+ monothan normal—did not reach statistical significance
cytic cells were evident in the iris on day 11 in most
(Table 2). Part of this increase was the result of a
eyes (Table 3). Interestingly, clinically detectable dismarked rise in the number of round Ia+ cells, which
ease was evident in only one of these animals in whose
was particularly evident in the peripheral choroid
eye the ED1+ monocytic infiltrate was so severe that
(Fig. 4B, Table 2).
it formed a complete cellular "sheet" over the anterior iris surface (Fig. 5A). Such dense inflammatory
T Cells in the Choroid During the Prodromal
infiltrates precluded counting individual cells and preStage of Experimental Autoimmune
vented optimal immunostaining of the underlying iris.
Uveoretinitis
Occasionally, the mononuclear cell infiltrate was less
well
developed and involved only focal patches of ciliThe density of T cells in the normal choroid was ex2
ary
body
and iris vasculature and stroma (Fig. 5B).
tremely low (16 ± 7 cells/mm ). There was a marked
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Choroid Cell Density
Day 5
Day 7
Normal
Pertussis
EAU
c
591 (52)
122 (34)
465 (50)
4(1)
562 (37)
133 (26)
423 (48)
6(1)
659 (33)
40(3)
614 (22)
5(2)
521
65
451
5
c
723
350
342
31
647
268
339
40
807 (83)
140 (9)
630 (79)
37(6)
c
580 (26)
59 (12)
515 (18)
6(2)
505 (18)
56 (8)
442 (24)
7(2)
922 (45)
35 (18)
861 (20)
26(9)
c
746
136
571
39
674
157
456
61
c
(47)
(66)*
(40)
(7)
(38)
(15)
(32)
(6)
16(7)
0
0
16(7)
(38)
(26)*
(56)
(8)
(28)
(35)
(36)
(13)
71 (4)
0
15(1)
56(3)
1040
84
895
61
(83)
(24)
(115)
(15)
64 (18)
0
6(3)
58 (15)
Control
Control
EAU
Day
Control
EAU
Day 42
Control
670 (10)
48 (19)
618 (13)
4(2)
580 (69)
67 (13)
509 (55)
4(2)
625
48
574
3
(21)
(10)
(26)
(2)
573 (27)
117 (7)
450 (18)
6(3)
595 (35)
65 (29)
520 (24)
10(2)
565
126
436
3
(52)
(21)
(63)
(1)
744 (61)
147 (32)
578 (73)
19(7)
724
143
558
23
(31)
(28)
(30)
(10)
791
163
599
29
(152)
(5)
(123)
(4)
737
199
518
20
(204)
(32)
(130)
(4)
748
178
547
23
(51)
(21)
(47)
(2)
531
137
379
15
620
166
424
30
672 (14)
22 (11)
643 (13)
7(4)
798
32
746
20
(68)
(12)
(83)
(3)
864
53
801
10
(80)
(12)
(73)
(5)
893
34
838
21
(81)
(11)
(96)
(5)
777
57
710
10
(21)
(20)
(29)
(3)
625 (72)
28(5)
593 (76)
4 (2)
895
89
753
43
(48)
(19)
(50)
(12)
952
190
722
40
(128)
(36)
(87)
(5)
916
130
730
56
(42)
(24)
(64)
(4)
1035
126
841
68
(26)
(42)
(66)
(4)
1111
161
915
35
(11)
(19)
(14)
(1)
EAU
Day 9
(94)
(37)
(92)
(3)
57 (16)
0
6(2)
51 (15)
61 (18)
0
3(2)
58 (18)
59 (24)
0
7 (4)
52 (21)
50 (4)
0
7 (6)
43(7)
76 (10)
0
8(6)
68 (5)
mental autoimmune uveitis.
an cell density (±SEM).
sus normal.
sus normal.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017
1152
101
941
110
(106)
(39)
(46)
(2)
(192)
(43)
(139)
(53)
286 (161)
0
14(9)
272 (152)
EAU
(21)
(34)
(34)
(1)
624
56
565
3
(86)
(9)
(82)
(8)
667 (71)
137 (22)
515 (69)
15 (6)
595
122
461
12
694
52
632
10
(35)
(20)
(55)
(2)
664 (25)
44 (18)
616 (25)
4(1)
630
33
589
8
916
165
700
51
(91)
(15)
(84)
(21)
1233 (146)f
158 (29)
1038(144)
37 (8)
823
120
671
32
187 (54) t
0
16(3)
171 (56)
66
0
9
57
57 (3)
0
8(3)
49 (2)
535
75
457
3
C
Uveal Tract Immune Cells in Experimental Uveoretinitis
-at.
2203
marked ED1+ mononuclear cell infiltration of the iris
at day 11 also exhibited marked focal infiltrates of
small, round Ia+ cells throughout the iris stroma (Fig.
5C, Table 3). In addition, the sheet of mononuclear
cells that covered the anterior iris surface in one animal contained numerous round Ia+ cells. These are
most likely a mixture of Ia+ monocytes-exudate macrophages, activated T cells, and DC.
T cells are extremely rare in the normal iris (16
± 6 cells/mm^) (Fig. 5E). In the early stages of EAU
before disease onset (days 5, 7, and 9), there was no
quantitative change in T-cell numbers. However, by
day 11, disease onset was characterized by an influx
of small, round OX19-R73+ T cells throughout the
iris (Table 3, Fig. 5F). The density (196 ± 72 cells/
mm2) was evident as focal aggregates and as isolated
individual cells.
Immune Cells in the Iris in Late-Phase
Experimental Autoimmune Uveoretinitis
(Day 42)
The total number of Ia+ cells in the iris of die day
42 animals was approximately four times greater than
normal values (Table 3). Of special significance, however, was the presence of a distinct layer of extremely
dendriform Ia+ cells that appeared to be situated on
or close to the anterior iris surface (Fig. 5D). T cells
remained significandy elevated in the iris of day 42
animals (Table 3).
DISCUSSION
FIGURE4. Ia(OX6)+ cells in the central (A) and peripheral
(B) choroid in a day 11 experimental autoimmune uveoretinitis (EAU) Lewis rat. Note the increased number of predominantly round and pleomorphic cells close to vessel
walls. (C) Day 11 EAU choroid stained with pan-T-cell
marker (Tc). Note the exclusively small round cells evenly
distributed in the choroid. Magnifications: (A) X200; (B)
X80; (C) X200.
There was a slight decrease in the total density of
ED3 + iris macrophages in day 11 EAU animals (Table 3).
The network of Ia + DC in the iris was not markedly
altered in die early prodromal stages of EAU (days 5,
7, and 9) (Table 3). Those EAU animals that showed
Networks of DC are distributed ubiquitously in most
tissues and are particularly well developed and characterized in epithelial sites close to the external environment, such as skin, gut, and respiratory tract, where
they function as sentinels to trap and sample large
numbers of exogenous antigens.14 On migration to Tcell rich zones of draining lymphoid tissues, they mature into potent APCs and may present antigen to
naive T cells, thus initiating primary immune responses. In addition, DC are significantly more efficient APCs than B cells and macrophages in the induction of secondary immune responses.14 Dendritic cell
populations in the interstitium of nonrymphoid organs4445 are also likely to encounter altered self-antigens as a result of tissue injury, regeneration, and
repair.46
Many forms of endogenous intraocular inflammation are diought to have an autoimmune etiology,
and several autoantigens have been identified that can
induce symptoms similar to posterior uveitis. In these
models, immunization at distant sites with a potential
uveitogen leads to antigen-specific T-cell proliferation
in local lymph nodes. On entering the circulation, T
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017
ris Cell Density
Normal
634 (41)
c 361 (25)
hic 264 (29)
9 (2)
c
c
c
ic
Day 7
Day 5
603
399
187
17
(42)
(41)
(16)
(5)
Pertussis
EAU
533 (24)
203 (18)
299 (17)
31 (4)
552 (38)
113 (11)
419 (36)
20 (6)
468
131
317
19
(22)
(11)
(18)
(3)
496
107
371
18
(34)
(24)
(50)
(4)
514
125
365
24
(47)
(16)
(38)
(3)
406
106
288
12
(22)
(16)
(7)
(2)
360
153
165
43
356
110
229
17
383
72
269
42
(103)
(18)
(94)
(21)
331
89
208
35
(55)
(31)
(16)
(11)
260
83
162
15
(58)
(23)
(38)
(4)
392
136
233
23
(86)
(42)
(52)
(10)
(32)
(13)
(25)
(6)
(59)
(18)
(48)
(5)
Control
EAU
Day 9
Control
EAU
Day 11
Control
Day 42
EAU
Control
EAU
416 (72)
133 (30)
263 (47)
20 (1)
423 (132)
94 (51)
301 (87)
28 (10)
491 (56)
130 (35)
336 (30)
25 (4)
317 (77)
56 (4)
248 (72)
13 (2)
35
4
28
410
154
233
23
369
48
179
141
340
88
215
37
540
110
380
50
31
7
21
3
(42)
(19)
(40)
(13)
(112)
(26)
(68)
(60)
(54)
(18)
(45)
(4)
(101)
(45)
(75)
(10)
C
385 (29)
210 (8)
264 (35)
254 (44)
281 (66)
316 (85)
336 (62)
336 (115)
173 (89)
270 (67)
356 (52)
108 (17)
29 (3)
20 (4)
9 (6)
2 3 (12)
14 (5)
27 (4)
31 (18)
14 (10)
30 (15)
20 (7)
1
262 (24)
16 (3)
136 (9)
45 (1)
228 (37)
16 (9)
224 (45)
21 (5)
231 (45)
27 (13)
278 (84)
24 (3)
271 (75)
39 (10)
270 (79)
35 (18)
146 (75)
13 (6)
214 (60)
26 (6)
291 (55)
44 (9)
19
1
436
298
124
14
395
159
215
21
607
194
393
20
459
121
319
19
483
161
297
25
436
139
280
18
544
188
330
27
(129)
(15)
(110)
(6)
606
207
378
21
(31)
(27)
(30)
(7)
495
93
325
78
(162)
(51)
(156)
(46)
465
135
306
25
60
2
11
46
(23)
(2)
(9)
(12)
35 (11)
0
1 (1)
34 (11)
196
1
9
186
(72)
(1)
(6)
(68)
(43)
(28)
(19)
(4)
16 (6)
0
0
16
(27)
(18)
(16)
(4)
12 (5)
0
1 (1)
11 (5)
25
1
1
23
(106)
(30)
(94)
(10)
(9)
(1)
(1)
(8)
(48)
(24)
(24)
(1)
44 (13)
0
1 (1)
43 (13)
(104)
(33)
(5)
(16)
27 (13)
0
1 (1)
25 (14)
(63)
(23)
(34)
(8)
12 (3)
0
.1 (1)
10 (2)
mental autoimmune uveitis.
an cell density (±SEM).
sus normal.
rsus normal.
ersus normal.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017
(24)
(19)
(17)
(6)
41 (10)
0
2 (1)
40 (10)
1691
454
1191
46
(108)*
(56)
(78)
(9)f
152 (24)}
0
23 (7)
129 (29)
23
66
18
46
1
2
2
Uveal Tract Immune Cells in Experimental Uveoretinitis
Tc
FIGURES. Light micrographs of immunostained rat iris wholemounts in experimental autoimmune uveoretinitis (EAU). (A) Low-power view of dense aggregates of EDI+ mononuclear
cells forming a cellular "sheet" that covered the anterior surface of the iris in a day 11 EAU
animal. (B) Focal aggregate of ED1+ cells in the iris in a day 11 EAU animal. (C) Highpower view of la stained iris from a day 11 EAU animal, illustrating the focal aggregation
of Ia+ cells and the predominantly rounded and pleomorphic shape of the cells. (D) Ia+
cells in the rat iris in the late phase of EAU. Note the increased density of the DC network
and, in particular, the highly dendriform cells in focus lying close to the iris surface. The
remainder of the DC population is out of the focal plane. (E) Normal rat eye stained with
the pan-T-cell (Tc) marker combination of mAbs OX19-R73. (F) T cells in day 11 EAU
animal. Magnifications: (A,B) X78; (C to F) X195.
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2205
2206
Investigative Ophthalmology 8c Visual Science, October 1996, Vol. 37, No. 11
cells may adhere to postcapillary venules in the retina
or uveal tract before extravasating into the surrounding tissue, where it is postulated they interact
with autoantigen in the context of MHC class II+ APC.
The precise nature of the APC in the eye in the EAU
model is subject to speculation.
The current study confirms previous evidence that
rich networks of DC exist in the iris, ciliary body,18"21
and choroid22 at comparable densities to other nonocular tissues.47 Recent evidence has demonstrated that
the functional capacity of DC isolated from anterior24
and posterior portions of the uveal tract25 are comparable to other DC, such as epidermal Langerhans cells.
This makes them likely APCs in the sensitizing and
inductive phases of endogenous uveitis. Furthermore,
because of their anatomic location, immediately on
either the internal or external interface of the bloodocular barrier (iris stroma and basal aspect of retinal
pigment epithelium, respectively), uveal tract DC are
probably exposed to retinal antigens. The mechanisms that modulate immune responses within the
normal eye by prevention or downregulation of unwanted antigen presentation of ocular-derived antigen
by the rich networks of intraocular DC are now under
critical investigation in many laboratories (see reviews23'48).
In light of the potential importance of uveal tract
DC to the pathogenesis of posterior uveitis, one of the
specific aims of the current study was to obtain data
on the dynamics of DC in the early and late phase of
uveitis using the EAU model. Although several studies
have reported MHC class II upregulation on retinal
pigment epithelium and retinal vascular endothelium
in EAU models,4'1"52 sympathetic ophthalmia, and uveitis,53 there have been only limited reports of MHC
class II expression on individual cells in the choroid or
retina. However, a recent semiquantitative histologic
study of adhesion molecules and MHC class II expression in melanin-associated, antigen-induced uveitis described an increase in MHC class II+ cells in the iris
and choroid during the onset and peak of the disease.54 Our study, with the advantage of wholemount
staining, has demonstrated obvious increases in the
density of MHC class II+ cells in the iris and choroid
in the early stages of EAU. Although some of the increase may be accounted for by MHC class II+ activated T cells or macrophages, it probably represents
enhanced recruitment of DC precursors from the circulation or an upregulation of la on previously Ia~ DC.
There is evidence that a population of DC precursors
(OX62'Ia) exists within the choroid22 and iris (data
not shown). These may provide a rapidly recruitable
source of DC in the event of local inflammatory episodes. The concurrent increase in density of Ia+ DC
and T-cell infiltration in the uveal tract would provide
the appropriate environment for local activation of
uveitogenic T cells. The crucial importance of the interaction between the T-cell receptor and peptidebearing MHC class II molecule in induction of autoimmune disease has underpinned the rationale of experimental attempts to disrupt the complex using anti-la
antibody therapy and, therefore, to ameliorate diseases such as EAU51'55 and experimental autoimmune
(allergic) encephalomyelitis (EAE) .r>(1 The partial success of such studies supports the concept that MHC
class II expression is involved in the induction and
perpetuation of EAU. There is evidence that EAE may
be transferred to naive animals by sensitized DC57;
however, similar studies have yet to be performed with
ocular autoimmune models.
Macrophages are long lived, functionally heterogeneous cells whose phenotype is determined by their
developmental stage, state of activity, and location.
This heterogeneity and associated variable expression
of surface and cytoplasmic markers is commonly used
for the characterization of various macrophage subpopulations.3640 The current study has revealed that
uveal tract macrophages are a heterogeneous population. For example, normal iris macrophages are
weakly ED3+, but choroidal macrophages are strongly
ED3+. This mAb recognizes sialoadhesins and is
weakly expressed on other resident tissue macrophages but is strongly expressed on "activated"
lymphoid macrophages, where it is postulated these
cells play an immunosuppressive role.40'58 The significance of the differential expression of ED3 between
the iris and choroidal macrophages is unclear.
Uveal tract macrophages differ radically in immunophenotype from the resident tissue macrophage
population within the retina (microglia), which are
ED2", ED3~, and Ia~ but weakly ED1+.20 Similar observations have been made in the brain and spinal cord,
in which meningeal macrophages differ widely in phenotype and function from microglia within the neural
parenchyma.28'59-W) Data on the phenotypic and morphologic changes in retinal microglia during EAU
would have complemented the current study. This was
not possible, however, because of difficulties in obtaining immunostaining throughout the entire thickness of retinal wholemounts that was sufficiendy reliable and consistent for quantitative analysis. The fixation and staining protocols used to preserve and
visualize macrophages in the uveal tract do not reveal
retinal microglia. Therefore, an investigation of these
cells in EAU would have to be undertaken as a distinct
exercise.
Our data suggest that immediately before fullscale inflammation, there is surprisingly little alteration in the resident iris and choroidal macrophages,
although the slighdy more pleomorphic shape may
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Uveal Tract Immune Cells in Experimental Uveoretinitis
2207
indicate evidence of motility and activation. Similarly,
the resident macrophages showed little alteration in
form during the late phase of EAU. Our data suggest
the majority of macrophages in choroidal lesions in
EAU are newly extravasated ED1 + monocytes-macrophages. This concurs with similar histologic observations in IRBP-induced EAU61 and in the experimental
melanin-protein-induced uveitis model. 10 The role of
infiltrating macrophages in the immunopathology of
EAE, both in directly mediating damage to the central
nervous system (CNS) and in attracting other cells
to lesions, is well accepted62'63 and is supported by
depletion studies in which marked suppression of the
clinical signs of disease have been obtained. 31 To date,
there are no comparable published reports of prevention of EAU using macrophage depletion.
8 days after onset65; in another nonquantitative
study,66 T-helper cells were noted in 4 of 8 eyes examined between days 21 and 31 after immunization. Evidence from EAE studies suggest few T cells remained
in the CNS after recovery, although the meninges
were not specifically studied.71 These authors proposed that the decline in T cells and macrophages in
the CNS in the recovery phase of EAE was caused by
apoptosis in situ.72 On the basis of newly emerging
evidence, it appears that similar mechanisms may operate in the normal eye, thus reducing the likelihood
of unwanted tissue damage. 73 The situation in the recovery phase of ocular inflammatory disease remains
to be elucidated.
The consistent identification of small numbers of
T cells (approximately 10 to 20 cells/mm 2 ) in the
normal iris and choroid adds support to the recently
advanced concept that the CNS is normally patrolled
by a small number of T cells.64 Characterization of Tcell subsets was not performed in the current investigation because of the number of mAbs investigated and
the limited amount of tissue from each eye. In previous immunohistochemical studies of T-cell subsets in
EAU6506 the ciliary body, followed closely by the iris,
was identified as the earliest site of cellular infiltration.65 Initially, our data appeared to suggest that the
choroid contained T-cell numbers similar to those in
the iris on day 11; however, in light of the fact that Tcell numbers were marginally elevated in the iris on
day 9 and monocytic infiltrate began earlier in the iris
than in the choroid, we support the contention that
inflammatory cell infiltration begins in the anterior
uvea before the posterior uveal tract. Increased T-cell
density in the choroid and iris in EAU was caused by
either continuous extravasation from the circulation,
local in situ proliferation, or both. Studies of BrdU
incorporation in EAE lesions suggests that T-cell proliferation in the CNS is minimal,67 leading to the suggestion that T cells enter an unresponsive state in the
CNS microenvironment. The iris and choroid, because of their anatomic homology, are more likely to
behave similarly to the leptomeninges and subarachnoid space, which are reported to act as important
sites of T-cell precursor proliferation and effector cell
selection in EAE.68'69 Recent studies have suggested
antigen nonspecific T cells have an important role in
amplification of the disease process in EAU.70 The
demonstration of persistently raised T-cell numbers in
the uveal tract 42 days after immunization (approximately 30 days after disease onset) and after subsidence of the active inflammatory phase of EAU has
not been described in previous studies. Other studies
have described T cells returning to near normal levels
autoimmune, choroid, dendritic cell, iris, macrophages, T
cell, uveitis
Key Words
Acknowledgments
The authors thank Julie Crewe for performing the doublecolor immunolabeling.
References
1. Forrester JV, Liversidge J, Towler H, McMenamin PG.
Comparison of clinical and experimental uveitis. Curr
Eye Res. 1990;9(suppl):75-84.
2. Forrester JV, Liversidge J, Dua HS, Dick A, Harper F,
McMenamin PG. Experimental autoimmune uveoretinitis: A model system for immunomodulation. Curr
Eye Res. 1992;ll(suppl):33-40.
3. Faure JP. Autoimmunity and the retina. Curr Top Eye
Res. 1980; 2:215-302.
4. McMenamin PG, Broekhuyse RM, Forrester JV. Ultrastructural pathology of experimental autoimmune
uveitis: A review. Micron. 1993; 24:521-546.
5. Nussenblatt RB. Experimental autoimmune uveitis:
Mechanisms of disease and clinical therapeutic indications. Invest Ophthalmol Vis Sri. 1991;32:3131-3141.
6. Caspi RR, Roberge FG, McAllister GG, et al. T cell
lines mediating experimental autoimmune uveoretinitis in the rat. J Immunol. 1986; 136:928-933.
7. Gery I, Mochizuki M, Nussenblatt RB. Retinal specific
antigens and immunopathogenic processes they provoke. Prog Ret Res. 1986;5:75-109.
8. de Kozak Y, Sakai J, Thillaye B, Faure JP. S antigeninduced experimental autoimmune uveoretinitis in
rats. Curr Eye Res. 1981; 1:327-337.
9. Broekhuyse RM, Kuhlmann ED, Winkens HJ, Van
Vugt AHM. Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis: I:
Induction by a detergent-insoluble, intrinsic protein
fraction of the retinal pigment epithelium. Exp Eye
Res. 1991;52:465-474.
10. Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune anterior uveitis (EAAU): II:
Dose-dependent induction and adoptive transfer us-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017
2208
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11
ing a melanin-bound antigen of the retinal pigment
epithelium. Exp Eye Res. 1992;55:401-411.
Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune anterior uveitis (EAAU): III: Induction by immunization with purified uveal and skin
melanins. Exp Eye Res. 1993;56:575-583.
Chan CC, Hikita N, Dastgheib K, Whitcup SM, Gery
I, Nussenblatt RB. Experimental melanin-protein-induced uveitis in the Lewis rat. Ophthalmology. 1994;
101:1275-1280.
Bora NS, Kim MC, Kabeer NH, et al. Experimental
autoimmune anterior uveitis: Induction with melaninassociated antigen from the iris and ciliary body. Invest
Ophthalmol Vis Sd. 1995; 36:1056-1066.
Steinman RM. The dendritic cell system and its role
in immunogenicity. Annu Rev Immunol. 1991; 9:271296.
Schwartz RH. T-lymphocyte recognition of antigen in
association with gene products of the major histocompatibility complex. Annu Rev Immunol. 1985; 3:237261.
Janeway CA. The role of CD4 in T-cell activation: Accessory molecule or co-receptor?. Immunol Today.
1989; 10:234-238.
Streilein JW, Wilbanks GA, Cousins SW. Immunoregulatory mechanisms of the eye. / Neuroimmuol. 1992;
39:185-200.
Knisely TL, Anderson TM, Sherwood ME, Flotte TJ,
Albert DM, Granstein RD. Morphologic and ultrastructural examination of I-A+ cells in the murine iris.
Invest Ophthalmol Vis Sd. 1991;32:2423-2431.
McMenamin PG, Holthouse I. Immunohistochemical
characterisation of dendritic cells and macrophages
in the aqueous outflow pathways of the rat eye. Exp
Eye Res. 1992;55:315-324.
McMenamin PG, Holthouse I, Holt PG. Class II MHC
(la) antigen-bearing dendritic cells within the iris and
ciliary body of the rat eye: Distribution, phenotype,
and relation to retinal microglia. Immunology. 1992;
77:385-393.
McMenamin PG, Crewe J, Morrison S, Holt PG. Immunomorphological studies of macrophages and MHC
class II-positive dendritic cells in the iris and ciliary
body of the rat, mouse and human eye. Invest Ophthalmol Vis Sd. 1994; 35:3234-3250.
Forrester JV, McMenamin PG, Holthouse I, Lumsden
L, Liversidge J. Localisation and characterization of
major histocompatibility complex class II-positive cells
in the posterior segment of die eye: Implications for
induction of autoimmune uveoretinitis. Invest Ophthalmol Vis Sd. 1994; 35:64-77.
McMenamin PG. Immunocompetent cells in the anterior segment. Prog Ret Eye Res. 1994; 13:555-589.
Steptoe RJ, Holt PG, McMenamin PG. Demonstration
of the immunostimulatory capacity of dendritic cells
isolated from the rat iris. Immunology. 1995; 85:630637.
Choudhury A, Palkanis VA, Bowers WE. Characterisation and functional activity of dendritic cells from rat
choroid. Exp Eye Res. 1994;59:297-304.
26. Perry VH, Gordon S. Macrophages and microglia in
the nervous system. Trends Neurosd. 1988; 11:273-277.
27. Hickey WF, Vass K, Lassmann H. Bone marrow-derived elements in the central nervous system: An immunohistochemical and ultrastructural survey of rat
chimeras. JNeuropathol Exp Neurol. 1992;51:246-256.
28. Perry VH. Macrophages and the Nervous System. Austin,
TX: RG Landes; 1994:28-42.
29. LoweJ, MacLennan KA, Powe DG, Pound JD, Palmer
JB. Microglial cells in human brain have phenotypic
characteristics related to possible function as dendritic
antigen presenting cells. J Pathol. 1989; 159:143-149.
30. Penfold PL, Provis JM, Liew SCK. Human retinal microglia express phenotypic characteristics in common
with dendritic antigen-presenting cells. / Neuroimmunol. 1993;45:183-192.
31. Huitinga I, van Rooijen N, de Groot CJA, Uitdehaag
BMJ, Dijkstra CD. Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination
of macrophages. J Exp Med. 1990; 172:1025-1033.
32. Janeway CA. Ligands for the T-cell receptor: Hard
times for avidity models. Immunol Today. 1995; 16:223225.
33. Greenwood J. The blood-retinal barrier in experimental autoimmune uveitis (EAU): A review. CurrEye
Res. 1992;ll(suppl):25-32.
34. Steptoe RJ, McMenamin PG, McMenamin CC. Choroidal mast cell dynamics during experimental autoimmune uveitis in rat strains of differing susceptibility.
Ocul Immunol Inflammation. 1994; 2:7-22.
35. de Kozak Y, Thillaye B, Renard G, Faure JP. Hyperacute form of experimental autoimmune uveoretinitis
in Lewis rats: Electron microscopic study. Albrecht von
Graefes Arch klin exp Ophthalmol. 1978;208:135-142.
36. Dijkstra CD, Dopp EA, van der Berg TK, Damoiseaux
JGMC. Monoclonal antibodies against rat macrophages. / Immunol Methods. 1994; 174:21 -23.
37. McMenamin PG, Crewe J. Endotoxin-induced uveitis:
Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body.
Invest Ophthalmol Vis Sd. 1995;36:1949-1959.
38. Claassen E, Alder LT, Adler FL. Double immunocytochemical staining for in situ study of allotype distribution during an anti-TNP immune response in chimeric rabbits. JHistochem Cytochem. 1986;34:989-994.
39. Gunderson HJG, Bendtsen TF, Korbo L, et al. Some
new, simple and efficient stereological methods and
their use in pathological research and diagnosis.
APMIS. 1988;96:379-394.
40. Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: Distinct macrophage subpopulations in the rat
recognised by monoclonal antibodies EDI, ED2 and
ED3. Immunology. 1985; 54:589-599.
41. Damoiseaux JG, Dopp EA, Neefjes JJ, Beelen RHJ,
Dijkstra CD. Heterogeneity of macrophages in die rat
evidenced by variability in determinants: Two new
anti-rat macrophage antibodies against a heterodimer
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017
2209
Uveal Tract Immune Cells in Experimental Uveoretinitis
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
of 160 and 95 kd (CD11/CD18). / Leukocyte Biol.
1989;46:556-564.
Damoiseaux JG, Dopp EA, Calame D, Chao D, MacPherson GG, Dijkstra CD. Rat macrophage lysosomal
membrane antigen recognized by monoclonal antibody EDI. Immunology. 1994;83:140-147.
McMaster WR, Williams AF. Identification of la glycoproteins in the rat thymus and purification from rat
spleen. EurJImmunol. 1979;9:426-433.
Hart DNJ, Fabre JW. Demonstration and characterization of la-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not
brain. J Exp Med. 1981; 154:347-361.
Spencer S, Fabre JW. Characterization of the tissue
macrophage and the interstitial dendritic cell as distinct leukocytes normally resident in the connective
tissue of the rat heart. JExpMed. 1990; 171:1841-1851.
Ibrahim MAA, Chain BM, Katz DR. The injured cell:
The role of the dendritic cell system as a sentinel
receptor pathway. Immunol Today. 1995; 16:181-186.
Bergstresser PR, Fletcher CR, Streilein JW. Surface
densities of Langerhans cells in relation to rodent
epidermal sites with special immunologic properties.
J Invest Dermatol. 1980; 74:77-80.
Forrester JV, Liversidge J, Kuppner M, Mesri M. Immunoregulation of uveoretinal inflammation. Prog Ret
Eye Res. 1995; 14:393-412.
Fujikawa LS, Chan CC, McAllister C, et al. Retinal
vascular endothelium expresses fibronectin and class
II histocompatibility complex antigens in experimental autoimmune uveitis. Cell Immunol. 1987; 106:139150.
Chan CC, Hooks JJ, Nussenblatt RB, Detrick B. Expression of la antigen on retinal pigment epithelium
in experimental autoimmune uveoretinitis. Curr Eye
Res. 1986;5:325-330.
Wetzig R, Hooks JJ, Percepo CM, Nussenblatt R, Chan
R, Detrick B. Anti-la antibody diminishes ocular inflammation in experimental autoimmune uveitis. Curr
Eye Res. 1988; 7:809-818.
Liversidge J, Thomson AW, Sewell HF, Forrester JV.
Cyclosporine A, experimental autoimmune uveitis,
and major histocompatibility class II antigen expression of cultured retinal pigment epithelial cells. Transplant Proc. 1988;20(suppl):163-169.
Chan CC, Detrick B, Nussenblatt R, Palestine A, Fujikawa LS, Hooks JJ. Expression of HLA-DR antigens on
RPE cells from patients with uveitis. Arch Ophthalmol.
1986; 104:725-729.
Kim MC, Kabeer NH, Tandhasetti MT, Kaplan HJ,
Bora NS. Immunohistochemical studies on melanin
associated antigen (MAA) induced experimental autoimmune anterior uveitis (EAAU). Curr Eye Res.
1995; 14:703-710.
Rao NA, Atalla L, Linker-Israeli M, et al. Suppression
of experimental uveitis in rats by anti-I-A antibodies.
Invest Ophthalmol Vis Sci. 1989; 30:2348-2355.
Smith RM, Morgan A, Wraith DC. Anti-class II MHC
antibodies prevent and treat EAE without APC depletion. Immunology. 1994; 83:1-8.
57. Knight SC, Mertin J, Stackpoole A, Clark J. Induction
of immune responses in vivo with small numbers of
veiled (dendritic) cells. Proc Natl Acad Sci USA. 1983;
80:6032-6035.
58. Dijkstra CD, Dopp EA, Huitinga I, Damoiseaux JGMC.
Macrophages in experimental autoimmune diseases
in the rat: A review. Curr Eye Res. 1992;ll(suppl):7579.
59. Sminia T, DeGroot CJA, Dijkstra CD, Koetsier JC, Polman CH. Macrophages in the central nervous system
of the rat. Immunobiobgy. 1987; 174:43-50.
60. Flaris NA, Densmore TL, Molleston MC, Hickey WF.
Characterization of microglia and macrophages in the
central nervous system of rats: Definition of the differential expression of molecules using standard and
novel monoclonal antibodies in normal CNS and in
four models of parenchymal reaction. Glia. 1993;
7:34-40.
61. Harper FH, Liversidge J, Thompson AW, Forrester JV.
Interphotoreceptor binding protein induced experimental autoimmune uveitis: An immunophenotypic
analysis using alkaline phosphatase anti-alkaline phosphatase staining, dual immunoflourescence and confocal microscopy. Curr Eye Res. 1992;ll(suppl):129134.
62. Polman CH, Dijkstra CD, Sminia T, Koetsier JC. Immunohistological analysis of macrophages in the central nervous system of Lewis rats with acute experimental allergic encephalomyelitis. J Neuroimviunol. 1986;
11:215-222.
63. Bauer J, Sminia T, Wouterlood FG, Dijkstra CD.
Phagocytic activity of macrophages and microglial
cells during the course of acute and chronic relapsing
experimental autoimmune encephalomyelitis. / NeurosdRes. 1994; 38:365-375.
64. Lassmann H, Schmied M, Vass K, Hickey WF. Bone
marrow derived elements and resident microglia in
brain inflammation. Glia. 1993;7:19-24.
65. Brown EC, Kasp E, Dumonde DC. Morphometric analysis of T lymphocyte comartmentation in experimental autoimmune uveoretinitis. Clin Exp Immunol.
1989; 77:422-427.
66. Chan CC, Mochizuki M, Nussenblatt RB, et al. T lymphocyte subsets in experimental autoimmune uveitis.
Clin Immunol Immunopathol. 1985;35:103-110.
67. Ohmori K, Hong Y, Fujiwara M, Matsumoto Y. In situ
demonstration of proliferating cells in the rat central
nervous system during experimental autoimmune encephalomyelitis: Evidence suggesting that most infiltrating T cells do not proliferate in the target organ.
Lab Invest. 1992;66:54-62.
68. Tsuchida M, Hanawa H, Hirahara H, Watanabe H,
Matsumoto Y, Sekikawa H. Identification of CD4~
CD8~ a/3 T cells in the subarachnoid space of rats
with experimental autoimmune encephalomyelitis: A
possible route by which effector cells invade the lesions. Immunology. 1994; 81:420-427.
69. Shin T, Kojima T, Tanuma N, Ishihara Y, Matsumoto
Y. The subarachnoid space as a site for precursor T
cell proliferation and effector T cell selection in ex-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017
2210
Investigative Ophthalmology & Visual Science, October 1996, Vol. 37, No. 11
perimental autoimmune encephalomyelitis./A/ieuroiramunol. 1995;56:171-177.
70. Caspi RR, Chan CC, Fujino Y, et al. Recruitment of
antigen-nonspecific cells plays a pivotal role in the
pathogenesis of a T cell-mediated organ-specific autoimmune disease, experimental autoimmune uveoretinitis. J Neuroimmunol. 1993; 47:177-188.
71. McCombe PA, Fordyce BW, de Jersey J, Yoong G,
Pender MP. Expression of CD45RC and la antigen
in the spinal cord in acute experimental allergic
encephalomyelitis: An immunocytochemical and
flow cytometric study. / Neurol Set. 1992; 113:177—
186.
72. Pender MP, Nguyen KB, McCombe PA, Kerr JFR.
Apoptosis in the nervous system in experimental allergic encephalomyelitis. J Neurol Sci. 1991; 104:81-87.
73. Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science. 1995; 270:11891192.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933414/ on 05/10/2017