Investigative Ophthalmology & Visual Science, Vol. 31, No. 7, July 1990
Copyright © Association for Research in Vision and Ophthalmology
ImmunohistochemicQl Localization of
Cathepsin D in Ocular Tissues
Takahiko Yamada, Saroshi Horo, ond Mokoro Tamai
Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues.
In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin
D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the
cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented
and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and
sphincter and dilator muscles of the iris; Miiller cells; ganglion cells and pigment epithelium of the
retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a
high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in
retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic
degradation of rod outer segments. Invest Ophthalmol Vis Sci 31:1217-1223, 1990
Cathepsin D [EC3.4.23.5] is a major acid protease
of lysosomal enzymes and is believed to play an important role in the catabolism of protein in various
tissues. Biochemical methods have demonstrated
that some ocular tissues such as retinal pigment epithelium (RPE), iris and ciliary body, choroid, and
corneal endothelium have a high activity of cathepsin
D.1'2 The highest activity of cathepsin D in ocular
tissues has been identified in RPE.13 The outer segments of mature rods continually undergo renewal,4
and the pigment epithelium is responsible for removing the discs of the terminal rod outer segments
(ROS).5'6 It has been pointed out that the pigment
epithelial cells must have a highly developed phagolysosomal system that is capable of continuously digesting large amounts of ROS.7 Results of in vitro
studies have revealed that cathepsin D degrades proteins of ROS8'9 and catalyzes the degradation of
highly purified rhodopsin to a glycopeptide.10" It is
probable, therefore, that cathepsin D contributes to
the digestion of ROS in RPE.
The purpose of the current study was to investigate
the immunohistochemical localization of cathepsin
D in ocular tissues. We sought to determine which
ocular cells have cathepsin D and to reveal the inFrom the Department of Ophthalmology, Tohoku University
School of Medicine, Sendai, Japan.
Submitted for publication: August 16, 1989; accepted November
20, 1989.
Reprint requests: Takahiko Yamada, MD, Department of Ophthalmology, Tohoku University School of Medicine, 1-1, Seiryomachi, Aoba-ku, Sendai, Miyagi 980, Japan.
tracellular distribution of cathepsin D, especially
in RPE.
Materials and Methods
The care and treatment of animals in this investigation were in compliance with the ARVO Resolution on the Use of Animals in Research.
Preparation of Cathepsin D
Cathepsin D from bovine spleen was purchased
from Sigma (St. Louis, MO) and used as a source of
antigen. Purified cathepsin D and crude extract from
bovine RPE were prepared as described previously.10
Preparation of Antibody
Purified cathepsin D from bovine RPE formed a
single band at 45.0 kD (Fig. 1, lane 1) and the purchased cathepsin D from bovine spleen formed three
bands (Fig. 1, lane 2) in the gel of sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis. We
retrieved the protein of cathepsin D from bovine
spleen at 45.0 kD, and extracted the protein from the
gel with LKB 2014 Extraphor Electrophoretic Concentrator (LKB, Bromma, Sweden). Rabbit antiserum to cathepsin D was made with this electrophoretically purified cathepsin D from bovine spleen. We
injected 150 /*g purified cathepsin D from bovine
spleen that was mixed with the same volume of
Freund complete adjuvant into the subcutaneous
space of the back of a 2-kg New Zealand White rabbit
four times every 3 weeks.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1990
2
— 92.5 K
— 66.2 K
—45.0 K
— 31 .OK
.21.5 K
—14.4 K
Fig. I. SDS-polyacrylamide gel electrophoresis of cathepsin D.
Running gel is 10% polyacrylamide (Coomassie blue stain). Lane 1,
purified cathepsin D from bovine RPE (2 ^tg/well); lane 2, cathepsin D from bovine spleen (6 ^g/well); lane 3, molecular weight
standard (4 ^g/well).
Vol. 31
phoresis in 10% polyacrylamide gel for 40 min by the
method of Laemmli.13 Proteins in the gel were electroblotted onto a nitrocellulose paper using Towbin
and co-workers' buffer system14 for 1 hr. A nitrocellulose paper was incubated for 1 hr at room temperature in rabbit anticathepsin D antiserum that was
diluted 1:100. A control paper was incubated in an
identical manner in normal rabbit serum. An electroblotted paper was then incubated for 2 hr at room
temperature in secondary antibody, peroxidase-labeled goat anti-rabbit IgG diluted 1:200, and the enzyme-conjugated complex was visualized with
4-chloro-l-naphthol in 0.005% hydrogen peroxide
(Fig. 3).
Immunostaining: Immunohistochemical evaluation of this antiserum was performed with bovine
spleen cells. The RPE from bovine, rat, and human
eyes were also examined immunohistochemically
with the following method.
Immunohistochemical Evaluation
Bovine eyes and spleen were obtained from the
local slaughterhouse. Rat eyes were obtained from
28-day-old Royal College of Surgeons (RCS)-rdy+
rats that had been raised from birth in a 12-hr
light:dark cycle and killed 1 hr after the onset of light.
Since RCS-rdy+ rat is the congenic control to the
Enzyme-Linked Immunosorbent Assay (ELISA)
The titer of antiserum to cathepsin D from bovine
spleen was examined with use of ELISA. Wells were
coated with cathepsin D from bovine spleen (1 jxgf
well) or bovine serum albumin (BSA; 1 /ig/well). As
the blocking solution, 15% fetal calf serum (FCS),
with 1% BSA-0.14 M NaCl-0.01 M phosphate buffer
(PBS), pH 7.4, (150 ^I/well) was used. Wells were
incubated with serially diluted rabbit anticathepsin D
antiserum or normal rabbit serum with 1% BSA-PBS
(50 ^il/well). Thereafter, wells were incubated with
alkaline-phosphatase conjugated goat anti-rabbit IgG
F(ab')2 (TAGO, Burlingame, CA) diluted 1:750 with
1% BSA-PBS (50 /xl/well). Color was developed with
the use of 6 mM p-nitrophenylphosphate (100 fil/
well). The absorbance at 405 nm was read. The conditions of incubation and washing and the instruments were the same as described previously.12 Three
days after the last injection of cathepsin D, we observed a significant increase in the titer of antiserum
to cathepsin D from bovine spleen (Fig. 2).
Specificity of Antibody
Immunoblotting: Purified cathepsin D and crude
extract from bovine RPE were processed by electro-
10
20
40
BO
180 320 640 12BO 2560 5120 tO24O 20480
SERUM DILUTION IfOLO)
Fig. 2. ELISA of rabbit anti-cathepsin D antiserum. Wells were
coated with cathepsin D from bovine spleen (filled squares, open
squares) and BSA (filled triangles, open triangles). Serially diluted
antiserum, rabbit anticathepsin D (filled squares, filled triangles),
and normal rabbit serum (open squares, open triangles) were used
as the first antibody.
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IMMUNOHI5TOCHEMICAL LOCALIZATION OF CATHEP5IN / Yomodo er ol
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1
2
1
1219
2
31.0 K —
21.5 K v
14.4 K—
B
Fig. 3. Immunoblotting (Western blotting) of purified cathepsin
D and crude extract from bovine RPE using rabbit anticathepsin D
antiserum. (A) Nitrocellulose strip was incubated with rabbit anticathepsin D antiserum. (B) Nitrocellulose strip was incubated with
normal rabbit serum as control. Lanes of each strip: 1, purified
cathepsin D, and 2, crude extract from bovine RPE. The positively
stained single band of each two lanes in the experimental strip
corresponded to the level of 45.0 kD.
RCS strain of pink-eyed rat that shows inherited retinal degeneration, the results of RCS-rdy+ rat in the
current study provide essential information for the
further studies on inherited retinal degenerations.
Fig. 5. Immunohistochemical localization of cathepsin D in RPE
of bovine (A, B), RCS-rdy+ rat (C, D), and human (E, F). Reaction
products are seen in the cytoplasm of RPE (arrows) (A, C, E). No
reaction product is seen in the controls (B, D, F). (A-F) Bar = 10
One human eye was obtained from a 40-yr-old
woman patient who had adenoid cystic carcinoma
that originated in the orbit.
Eyes were perforated at the pars plana and immersed in periodate-lysine-paraformaldehyde fixative (PLP)15 for 30 min. Next, they were bisected,
placed in PLP for 72 hr at 4°C, and routinely processed for paraffin embedding. Bovine spleen was
placed in PLP for 72 hr at 4°C and embedded in the
Fig. 4. Immunohistochemical localization of
cathepsin D in bovine
spleen. Cytoplasm of leukocytes is stained (arrows) (A).
No staining is observed in
control (B). (A, B) Bar = 10
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL 5CIENCE / July 1990
Vol. 01
B
Fig. 6. lmmunohistochemical localization of
cathepsin D in bovine cornea. Reaction products are
observed in the cytoplasm
of (A) epithelium (arrows)
and keratocytes (arrowheads), and (C) endothelium (arrows). There is no
staining in the controls (B,
D). (A-D)Bar = 10 /im.
r
same manner. Sections 4-^m thick were treated with
xylene and ethanol and washed with PBS and PBS
containing 0.05% Tween 20. Nonspecific peroxidase
activity was inhibited with a tO-min incubation in
0.3% H2O2-methanol at room temperature. Nonspecific reaction with second antibody was blocked with
a 15-min incubation in normal goat serum diluted
1:200 at room temperature. Thereafter, sections were
incubated in rabbit anticathepsin D antiserum diluted 1:500 for 12 hr at 4°C and in peroxidase-conjugated anti-rabbit IgG goat Fab diluted 1:50 (MBL,
Nagoya, Japan) for 30 min at room temperature serially. Antibodies were dissolved in PBS, and sections
were rinsed with PBS containing 0.05% Tween 20
between incubations. The enzyme-conjugated complex was visualized by incubation with 0.02% 3,3'diaminobenzidine-tetrahydrochloride (DAB; Sigma)
and 0.005% H2O2 in 0.05 M Tris-HCl buffer (pH 7.6)
for 2-6 min at room temperature. Counterstaining
was done with methyl green for 60 min at room temperature. Control sections were treated with normal
rabbit serum diluted 1:500 instead of rabbit anticathepsin D antiserum.
Results
Our results showed that rabbit anticathepsin D antiserum had affinity to cathepsin D not only from
bovine spleen but also from bovine R.PE (Fig. 3).
lmmunohistochemical methods showed that cells of
bovine spleen (Fig. 4) and of bovine, rat, and human
RPE (Fig. 5) were positively stained with this antiserum, indicating the reaction of this serum with
cathepsin D in these tissues.
In ocular tissues, immune reaction was observed in
the cytoplasm of the following cells: epithelium and
endothelium of the cornea; keratocytes (Fig. 6); pigmented and nonpigmented epithelium of the ciliary
body (Fig. 7); lens epithelium and cells at the lens
Fig. 7. lmmunohistochemical localization of
cathepsin D in bovine ciliary body. Reaction products are observed in (A) cytoplasm of nonpigmented
epithelium (large arrows)
and pigmented epithelium
(small arrow). Staining of
pigmented epithelium is
difficult to observe because
of pigment. No reaction
product is observed in control (B). (A, B) Bar = 10 jim.
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IMMUNOH15TOCHEMICAL LOCALIZATION OF CATHEPSIN / Yonnodo er a I
Fig. 8. Immunohistochemical localization of
cathepsin D in bovine lens.
(A) Cytoplasm of lens epithelium is stained (arrows).
Cortex and nucleus are detached. (B) There is no
staining in control. (A, B)
Bar = 10 nm.
cortex (Figs. 8, 9); epithelium and sphincter and dilator muscles of the iris (Fig. 9); Miiller cells, ganglion
cells and RPE of the retina (Fig. 10); and endothelium of vessels in various ocular tissues. The staining
pattern typical of the vascular endothelium was noticed in the choroid (Fig. 10).
In the retina, footplates (Fig. 10A) and processes
(Fig. 1OC) of Miiller cells also were positively stained.
Although most ganglion cells were stained strongly
(Fig. IOC), weakly stained ganglion cells were seen
occasionally. Immunoreactive cathepsin D was
found in relatively apical portions of the cytoplasm of
RPE (Fig. 10E). Two types of staining were observed
in RPE. One was 3-5 /im in size and round or oval,
and another was 1-2 fitn in size and round
(Fig. 10E).
There were no significant differences in the results
among bovine, rat, and human ocular tissues. Brown
reaction products of DAB, however, could not clearly
be identified in pigment-laden cells such as the pigmented epithelium of iris and ciliary body, RPE, and
melanocytes of bovine and human eyes. The cortex
of bovine lens could not be examined because of its
detachment from the slide glass.
Discussion
The distribution of cathepsin D in ocular tissues
has been studied with the use of biochemical
1221
B
\
methods.12 These procedures, however, cannot detail
the cellular distribution of this protease. Histochemical methods using enzymatic reaction in tissues can
reveal the histologic distribution of enzymes clearly,
and many reports have been prepared on the distribution of lysosomal enzymes other than cathepsin D
in the cornea,1617 RPE17"20 and various ocular tissues.17 Because the specificity of enzyme products in
the enzymatic reaction method is problematic, we
used immunohistochemical methods to investigate
the histologic distribution of cathepsin D in detail. To
our knowledge, this is the first report on the immunohistochemical localization of cathepsin D in whole
ocular tissues. Positively stained ocular tissues in this
study were believed to have a high activity of protein
catabolism.
In the cornea, reaction products were recognized in
the cytoplasm of epithelium, keratocytes, and endothelium (Fig. 6). In cases in which there were alkali
burns of the rabbit cornea, the distribution of lysosomal enzymes was changed in accordance with the
healing process in both traumatized and nontraumatized areas.16 Cathepsin D in the cornea might have
played a role in the healing process of the damaged
cornea. Corneal endothelial cells phagocytize melanin granules degraded by their lysosomes under certain pathologic conditions.21 An extralysosomal release of lysosomal enzymes may account for the de-
Fig. 9. Immunohistochemical localization of
cathepsin D in RCS-rdy+ rat
iris and Jens. Reaction products are observed in (A) cytoplasm of epithelium
(small arrowheads) and
sphincter (medium-size arrowheads) and dilator (large
arrowheads) muscles of the
iris and epithelium (large
arrows) and cortex (small
arrows) of the lens. (B) No
reaction product is observed
in control. (A, B) Bar = 10
B
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INVESTIGATIVE OPHTHALMOLOGY & VI5UAL 5CIENCE / July 1990
Fig. 10. Immunohistochemical localization of cathepsin D in
bovine retina and choroid. Reaction products are seen in (A) footplates of Miiller cells (small arrows) and cytoplasm of ganglion cells
(large arrows). (C) Positively stained cytoplasm of Miiller cells often
is observed in the inner nuclear layer (small arrows), in the outer
nuclear layer (large arrows), and processes near the outer limiting
membrane (arrowhead). (E) In RPE, numerous round and oval
areas are stained in the cytoplasm. The large areas (large arrows) are
3-5 nm and the small areas (small arrows) are 1-2 nm. The endothelium of the choroidal vessels is positively stained (arrowheads).
No control sections (B, D, F) are stained. (A-F) Bar = 10 nm.
generation of the endothelial cells in some pathologic
conditions.22 The high activity of cathepsin D in the
endothelium has already been demonstrated with
biochemical methods.2 It appears, therefore, that
cathepsin D in the endothelium might have participated in the digestion of macromolecules by the endothelium or in the pathogenesis of the endothelial
degeneration under certain conditions. Our current
results with the corneal endothelium also support
such conclusions.
It has been reported that under pathologic conditions the ciliary epithelium shows phagocytosis of the
foreign body that is associated with increased lysosomal enzyme activity.23 Ciliary epithelium has an
abundance of organelles, because it has an important
function in the production of aqueous humor. Cathepsin D in the ciliary epithelium probably contrib-
Vol. 01
utes to the degradation of phagosomes under pathologic conditions and of autophagosomes of organelles.
The cellular distribution of cathepsin D in lens has
been shown, we believe, for the first time in this
study. Biochemically, cathepsin D could not be detected in whole lens, although the lens epithelium
demonstrates lysosomal enzyme activity.24 Cathepsin
D in the lens epithelium and cortex possibly plays
some role in the digestion of organelles of cells in
these areas.
In the iris, sphincter and dilator muscles have an
abundance of organelles, because they have an essential function in the pupillary reflex. Cathepsin D in
these cells probably works in connection with the digestion of organelles.
Biochemical methods have demonstrated that the
activity of cathepsin D is extremely low in neural
retina compared with RPE.1 The cytoplasm of Miiller
cells and ganglion cells was, however, positively
stained. Cathepsin D in Miiller cells seems to digest
organelles or endosomes. Histochemical evaluation
has shown that ganglion cells have arylsulfatase activity.17 Lysosomal enzymes, including cathepsin D,
supposedly catalyze the degradation of macromolecules in ganglion cells cooperatively.
The intracellular distribution of reaction products
in RPE were readily examined in bovine nonpigmented RPE (Fig. 9E). Large (3-5-^tm) and small
(1-2-^m) areas of staining were observed in cytoplasm, and in relatively apical cytoplasm. The large
areas of staining seemed to represent phagolysosomes, whereas the small ones appeared to be lysosomes. This finding shows that cathepsin D localizes
close to ROS in RPE. To our knowledge, these data
have been shown for the first time in the current
study. According to these data, we believe that ROS
and rhodopsin in RPE are probably degraded by
cathepsin D in vivo.
Vascular endothelium in ocular tissues showed
consistently immunoreactive cathepsin D. In these
cells, cathepsin D seemed to contribute to the degradation of autophagosomes of organelles and of internalized macromolecules.
It has been reported that under physiologic conditions lysosomal enzymes relate to the aging of RPE
and Bruch's membrane20 and the phagolysosomal
system in RPE.25 There are also reports of the close
relationship between lysosomal enzymes and etiologies of pathologic conditions, such as alkali burns,16
heat damage,26 ischemia,27 inherited retinal dystrophy in RCS rats,18 and retinal dystrophy in spontaneously dystrophic rat.28 The current study, we hope,
will aid other research in dealing with the role of
lysosomal enzymes in such conditions.
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IMMUNOHISTOCHEMICAL LOCALIZATION OF CATHEPSIN / Yomada er ol
Key words: cathepsin D, immunohistochemistry, retinal
pigment epithelium, phagolysosomal system, RCS-rdy+ rat
Acknowledgments
The authors would like to express their thanks to Drs.
Masahisa Kyogoku and Takashi Sawai of the Department
of Pathology, and Dr. Sei-chi Ishiguro of the Department of
Ophthalmology, Tohoku University School of Medicine,
for their excellent suggestions and discussions.
15.
16.
17.
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