Abstract
purpose. The present study focuses on the role of CD81, the target of the
anti-proliferative antibody (TAPA), in the regulation of the growth of
retinal pigment epithelium (RPE).
methods. RPE of 8-day-old rat pups was cultured. The level of CD81 in the
cultures was defined by immunoblot methods, and the distribution of the
protein was examined using indirect immunohistochemical methods. In
addition, the effects of the antibody binding were tested in culture.
results. CD81 was found in all layers of the normal retina with a distinct
absence of labeling in the inner and outer segments of the
photoreceptors. Based on the authors’ original immunohistochemical
analysis, it was difficult to determine whether CD81 was
expressed by RPE. By examining cultures of RPE it was demonstrated that
CD81 was expressed on the surface of these cells and that it was
concentrated at regions of cell–cell contact. Indirect
immunohistochemical methods using a peroxidase-labeled secondary
antibody in albino mice revealed heavy labeling of the RPE in the
intact eye. When the AMP1 antibody (directed against the large
extracellular loop of CD81) was added to cultured RPE, the mitotic
activity of the cells was depressed.
conclusions. CD81 was found in the normal rat retina. Previous studies demonstrated
that CD81 was expressed in retinal glia, the Müller cells that
span the thickness of the retina, and astrocytes found in the ganglion
cell layer. The present study demonstrated that CD81 was also expressed
by RPE. The dramatic effects of the AMP1 antibody and the location of
CD81 at regions of cell–cell contact support the hypothesis that this
molecule is part of a molecular switch controlling contact
inhibition.
The response of the mammalian retina to injury is similar
to that occurring in other parts of the central nervous system. Like
the brain and spinal cord, the retina contains glial cells (astrocytes
in the ganglion cell layer, and Müller cells that span the
cellular layers) which display a reactive response after
injury.
1 2 The astrocytes and Müller cells can
hypertrophy and increase the expression of the intermediate filament
protein, glial fibrillary acidic protein (GFAP).
3 4 5 6 The
retinal glia and retinal pigment epithelium (RPE) can proliferate after
injury. For example, when the retina becomes detached, two distinct
sets of processes can occur. First, the Müller cells can send
their processes into the subretinal spaces between the photoreceptors
and RPE causing a glial scar.
7 This scar appears to
contribute to the absence of retinal reattachment and the death of
photoreceptors.
8 Second, the retinal glial cells and
pigment epithelium can also migrate into the vitreal space,
proliferate, and form cellular membranes.
9 This response
is known as proliferative vitreoretinopathy, and when these cellular
membranes contract, the retina can detach.
10 Proliferation
of non-neuronal cells in the retina is a common and deleterious feature
of both disease and injury, including diabetes, retinal
detachment,
7 photocoagulation,
5 proliferative
vitreoretinopathy,
1 10 and mechanical
injury.
6 11 Thus, reactive glial responses can have
serious consequences, potentially resulting in the loss of sight.
Understanding molecular mechanisms associated with these changes may
provide insights into interventions, which may stop or reverse the
detrimental effects of reactive gliosis and cellular proliferation in
the retina. The present proposal focuses on the role of CD81,
previously known as the target of the anti-proliferative antibody
(TAPA). CD81 is a member of the tetraspanin superfamily that consists
of an increasing number of members (CD9, CD37, CD53, CD63, TAPA-1[
CD81], CO-029, R2, CD82 [KAI1], CD151, Tspan 1–6, NAG-2, late
bloomer, and NET1–7).
12 13 14 15 16 17 18 19 20 21 As a family, the tetraspanins
appear to be part of a molecular complex
13 22 23 24 linking
cell–cell contact to changes in cellular behavior. In the present
study we examine the distribution of CD81 in RPE and define its role in
the proliferation of these retinal cells.
Indirect immunohistochemical methods were used to define the
cellular localization of CD81 in cultured cells, sections of retina and
dissected sheets of RPE. Cultured RPE cells were produced as described.
For sections of retina, both Long-Evans (two adult female rats) and
Sprague-Dawley (two male) rats were used. Adult rats were anesthetized
with a mixture of xylazine (13 mg/kg) and ketamine (87 mg/kg) which was
administered by intraperitoneal injection. The rats were perfused
through the heart with a solution of 0.1 M PBS (pH 7.5) followed by 4%
paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.5). All the
protocols used in this study were approved by the Animal Care and Use
Committee of the University of Tennessee, Memphis, and conformed to the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research. After the eyes were removed from the skull, the cornea was
dissected from the globe, and the lens was removed. The eyes were
placed in a 30% sucrose solution for at least 2 days. Cryostat
sections were taken at 30 μm (Reichert Histostat, Buffalo, NY).
In general the immunohistochemical methods used were similar, with only
minor variations depending on the specific application. The sections
were blocked, for 2 hours at room temperature, with 4% BSA (Sigma, St.
Louis, MO) in 0.2 M borate-buffered saline (BBS, pH 8.4). The sections
were then incubated in the monoclonal antibody AMP1
27 at a
dilution of approximately 20 μg/mL of 0.2 M BBS with 1% BSA
overnight at 4°C. Sections were washed three times for 10 minutes
each in 0.2 M BBS and incubated with peroxidase-conjugated goat
anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove,
PA) at a dilution of 1:250 in BBS for 2 hours at room temperature.
Sections were washed in BBS followed by 0.1 M PBS, three times for 10
minutes each, and incubated in a solution containing 25 mg of
3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) per 50 mL
phosphate buffer (pH 7.2) and 200 μL 3% hydrogen peroxide for 15 to
30 minutes at room temperature. When staining cultures of RPE or
dissected sheets of RPE a fluorescence-labeled secondary antibody was
used (Jackson ImmunoResearch Laboratories).
For electron microscopic examination, the dissected RPE was rinsed in
PBS and stained with the AMP1 antibody, followed by a conjugated goat
anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.). After the
cells were reacted with DAB, they were postfixed in 1% osmium
tetroxide for 1 hour at room temperature. The cells were rinsed in PBS,
dehydrated, and infiltrated with Spurr embedding media. Silver to gold
sections were obtained on a microtome (Ultracut E; Reichert Histostat)
and the sections were examined using an electron microscope (2000EX;
JEOL, Tokyo Japan).