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1 From the Department of Ophthalmology, Kurume University School of Medicine, Kurume, Japan; the 2 Department of Ophthalmology, Tokyo Medical and Dental University School of Medicine, Japan; and the 3 Department of Pathology, Kurume University School of Medicine, Kurume, Japan.
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Abstract |
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METHODS. Male inbred Lewis rats were immunized with S-antigen (40 µg/rat), and eyes were examined to detect apoptotic cells on days 1, 4, 8, and 10 post-immunization and days 0, 2, 4, 6, and 8 after the onset of EAU. The clinical and pathologic scores were used for estimating EAU. Apoptotic cells were analyzed by TdT-mediated dUTP nick-end labeling, electron microscopic and immunohistologic examinations, and agarose gel electrophoresis. The anti–rat Fas and anti–rat FasL antibodies were used to examine the expression of Fas and FasL.
RESULTS. Apoptotic cells were detected in the infiltrating cells in the aqueous humor, the vitreous body, the iris-ciliary body, and the retina. Apoptotic cells were observed as early as the day of EAU onset and reached a peak on day 2 after the disease onset. Fas and FasL were expressed on the infiltrating cells in the aqueous humor and the vitreous. FasL was expressed on resident cells of the ciliary body. The kinetics of the expression of FasL corresponded with the kinetics of apoptotic cells.
CONCLUSIONS. Fas–FasL–mediated apoptosis is considered to occur in the eye with EAU and plays a role in the immunopathogenic mechanisms to eliminate ocular infiltrating cells, thereby downregulating the inflammatory processes.
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Introduction |
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and interleukin (IL)-2,
whereas Th2 cells produce IL-4 and IL-10. Recent studies have shown
that Th1 responses are responsible for the onset of EAU in rats,
whereas Th2 responses downregulate the disease.3
However,
it has not yet been clarified how ocular infiltrating cells are
eliminated from the eye. Apoptosis is a mechanism that causes programmed cell death. Although apoptosis can be induced by a variety of signals, Fas–Fas ligand (FasL) are the most important signals to cause apoptosis.4 Interaction between Fas-positive cells and FasL molecules causes signal transduction in the Fas-positive cells, resulting in apoptosis of the cells. Fas–FasL interaction and apoptosis may occur in eyes with EAU and participate in the mechanism to downregulate the disease, but no studies have been carried out to examine the apoptosis and expression of Fas and FasL in EAU. The present study was, therefore, aimed at investigating the role of apoptosis and Fas–FasL in EAU.
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Methods |
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Immunization
S-antigen was prepared from bovine retinas according to the
methods described by Fujino et al.5
The antigen was
emulsified (1:1, vol:vol) in complete Freund’s adjuvant (Difco,
Detroit, MI), containing Mycobacterium tuberculosis H37 RA (Difco)
at a concentration of 2.0 mg/ml. A total volume of 100 µl per rat,
containing 40 µg S-antigen, was injected into one hind footpad.
Evaluation of EAU
Eyes of S-antigen–immunized rats were clinically examined with a
slit-lamp microscope on days 1, 4, 8, and 10 post-immunization, and on
days 0, 2, 4, 6, and 8 after the onset of EAU. The onset of EAU was
confirmed when fibrins were detected in the anterior chamber. The
clinical score was graded into four categories from 0 (no inflammation)
to 3+ (severe inflammation with hypopyon). Eyes were enucleated under
general anesthesia with diethylether on the days described above, fixed
with 2.5% glutaraldehyde 4% formaldehyde solution, and embedded in
paraffin. Sections cut at 4 µm were stained with hematoxylin and
eosin and examined with a light microscope. The EAU scores by
histologic examinations were graded from 0 (no inflammation) to 4+
(full thickness retinal damage in 
1/4 of the retina).
Terminal Deoxynucleotide Transferase–Mediated dUTP Nick End
Labeling Methods
In situ detection of apoptotic cells was conducted by terminal
deoxynucleotide transferase (TdT)–mediated dUTP nick end labeling
(TUNEL) methods using a commercially available kit (Apop Tag In Situ
Apoptosis Detection Kit; Oncor, Gaithersburg, MD), according to the
manufacturer’s protocol. Briefly, eyes of S-antigen–immunized rats
were fixed with a 2.5% glutaraldehyde 4% formaldehyde solution and
embedded in paraffin. Sections cut at 4 µm were deparaffinized and
stripped of proteins by incubation with 20 µg/ml proteinase K for 15
minutes at room temperature. Endogenous peroxidase was inactivated by
immersion in phosphate-buffered saline (PBS) containing 2%
H2O2 for 5 minutes at room
temperature. Residues of digoxigenin-nucleotide were catalytically
added to the DNA by TdT, an enzyme that catalyzes a
template-independent addition of deoxyribonucleotide triphosphate to
the 3'-OH ends of DNA. The reaction was conducted at 37°C for 1 hour.
The color reaction was visualized by peroxidase-conjugated
anti-digoxigenin antibody and 3,3'-diaminobenzidine (DAB). The sections
were counterstained by 0.5% methyl green. Distilled water was
substituted for TdT enzyme for the negative controls.
Electron Microscopic Examinations
A small aliquot of aqueous humor was centrifuged at 1500 rpm
(380g) for 5 minutes, and the pellets were fixed with 2.5%
glutaraldehyde for 1 hour at room temperature. The pellets were washed
by cacodylate buffer to remove the excess of glutaraldehyde and
postfixed with 1% OsO4 for 1 hour at 4°C. The
pellets were washed by cacodylate buffer again, embedded in stable
gels, centrifuged at 380g for 5 minutes, diced into 2-mm
cubes, and dehydrated by ethanol (50%, 70%, 90%, and 100%). The
cubes were immersed in 100% ethanol with epoxy resin (1:1, vol:vol)
for 12 hours at room temperature. They were then embedded in 100%
epoxy resin for 6 hours at room temperature and for 30 hours at 60°C.
The cubes were cut in thin sections (80 nm), placed on grids to be
stained with uranyl acetate and lead citrate, and studied by
transmission electron microscope.
DNA Gel Electrophoresis
The fragmentation of the nucleus of infiltrating cells in the
aqueous humor and the vitreous was determined by DNA gel
electrophoresis using a Apoptosis Ladder Detection Kit (Wako, Osaka,
Japan). A small aliquot of aqueous humor was collected from eyes of
S-antigen–immunized rats using a 30-gauge needle. A total of 1 x
106 cells was used for DNA gel electrophoresis
according to the manufacturer’s protocol. Briefly, the cells were
collected in a microcentrifuge tube by a brief centrifugation,
discarding the supernatant. Enzyme reaction solution, RNase solution,
enzyme activator solution, and protein digestion enzyme solution were
added to the tube and incubated at 50°C for 30 minutes. DNA
extraction solution was added to the tube and mixed well. Isopropanol
was added and left at room temperature for 15 minutes. The sample was
centrifuged at 10,000g at room temperature for 10 minutes
and decanted. Seventy percent ethanol was added and centrifuged at
10,000g at room temperature for 5 minutes and decanted. DNA
samples were extracted. The samples were electrophoresed in a 1.5%
agarose gel and visualized by SYBR green I.
Detection of Fas-Positive Cells and FasL-Positive Cells
The expression of Fas was examined by the
streptavidin-biotin peroxidase method using rabbit anti-mouse
Fas polyclonal antibodies (Wako) as the primary antibody. The
expression of FasL was examined by the same technique as Fas, but
rabbit anti–rat FasL polyclonal antibodies (Wako) were used as the
primary antibody. Eyes were enucleated under general anesthesia with
diethylether, fixed with 0.5% Zn acetate 0.5% ZnCl in Tris–Ca
acetate buffer,6
and embedded in paraffin.
Deparaffinized and rehydrated sections were immersed in 0.3%
H2O2 in methanol to block endogenous peroxidase
and were preincubated with 500 µg/ml goat IgG and 5% bovine serum
albumin (BSA) in PBS for 1 hour at room temperature to block
nonspecific binding of antibodies. The sections were reacted with the
primary antibodies (1:200) diluted with 1% BSA in PBS overnight at
4°C. Polyoxyethylene layryl ether (Brij35), a nonionic detergent, was
used to reduce nonspecific binding of antibodies. After washing in PBS
with 0.075% Brij35 three times for 15 minutes each time, the sections
were incubated with biotinylated goat anti-rabbit IgG (Histofine SAB-PO
[R]; Nichirei, Tokyo, Japan) for 2 hours at room temperature and
washed as described above. Peroxidase-conjugated streptavidin was
added, and the sections were washed again. The sites of peroxidase were
visualized by DAB and counterstained by 0.5% methyl green. Ovaries and
testes of naive healthy rats were used for positive control of Fas and
FasL.
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Results |
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Immunohistologic examination by TUNEL performed on day 2 after EAU onset demonstrated that TUNEL-positive cells were present in the aqueous humor, ciliary body, vitreous and retina and that the number of TUNEL-positive cells were higher in the aqueous humor and the vitreous than in the ciliary body and the retina (Fig. 1) . Electron microscopic examinations and DNA gel electrophoresis were performed to confirm that the TUNEL-positive cells were apoptotic cells. Electron microscopic examinations revealed that cells with highly condensed and aggregated chromatin in the nuclei were present in the aqueous humor (Fig. 2A ). The DNA ladder formation was clearly detected on day 2 after EAU onset using cells in the aqueous humor (Fig. 2B) . After confirming that the TUNEL-positive cells were apoptotic cells, kinetics of apoptotic cells were examined by TUNEL methods. No apoptotic cells were detected before the onset of EAU. Apoptotic cells were detected on the day of EAU onset and reached a peak on day 2 after EAU onset. The proportions of apoptotic cells in the infiltrating cells on day 2 after EAU onset were 12.4% in the aqueous humor and 10.0% in the vitreous. Examples of kinetics of apoptotic cells in the aqueous humor and the vitreous are shown in Figure 2C .
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Discussion |
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Apoptosis can be mediated by a variety of signals. One of the most important signals to cause apoptosis is Fas–FasL interaction.4 Current dogma is that ocular resident cells express FasL and that infiltrating T cells expressing Fas interact with FasL and then go into apoptosis.9 To investigate whether the Fas–FasL–mediated apoptosis takes place in EAU, the expression of Fas and FasL was studied in the eye with EAU by immunohistopathologic examinations. Fas expression was abundantly detected on infiltrating cells in the eye with EAU. Although in the present study FasL expression was hardly detected on ocular resident cells of naive rats, previous studies10 demonstrate significant expression of FasL on naive ocular resident cells, such as corneal endothelium. Therefore, it is considered that FasL is expressed on naive ocular resident cells, although the intensity of its expression might be weak. In contrast to naive ocular resident cells, ocular infiltrating cells abundantly expressed FasL in the present study. The previous studies together with the present study indicate two possible mechanisms by which T cells in the eye with EAU go into apoptosis: T cells go into apoptosis due to Fas–FasL interaction with other T cells and FasL expression on resident ocular cells is upregulated during inflammation followed by interaction with Fas-expressing T cells.
In conclusion, the present study suggests that Fas–FasL–mediated apoptosis occurs in the eye with EAU and that it plays a role in the immunopathogenic mechanisms to eliminate ocular infiltrating cells and downregulate the inflammation of EAU.
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Footnotes |
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Commercial relationships policy: N.
Corresponding author: Manabu Mochizuki, Department of Ophthalmology, Tokyo Medical and Dental University School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan. m.manabu.oph{at}med.tmd.ac.jp
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) and
the vitreous (
). *P < 0.05,
**P < 0.01. Error bars represent SD.
