Investigative Ophthalmology & Visual Science, Vol. 32, No. 5, April 1991
Copyright © Association for Research in Vision and Ophthalmology
Elastic Fiber Components and Protease
Inhibitors in Pinguecula
Zong-Yi Li, Robert N. Wallace, Barbara W. Srreeren, Bruce L. Kunrz, and Anthony J. Dark
The nature of the abnormal elastotic materials seen in pingueculae and their insensitivity to elastase are
poorly understood. The authors investigated their composition by immunoelectron microscopy using
antibodies to elastic fiber components, serum and tissue components known to be associated with
elastosis in other sites. The abnormal elastic fibers showed labeling for elastin, microfibrillar protein,
and amyloid P where these components never co-localize normally, indicating the fibers are not simply
immature but aberrant in organization. There was mild positivity for the serum protease inhibitor
alpha-1 antitrypsin at the edges of the abnormal elastic tissue and marked positivity for lysozyme. The
more superficial region of pingueculae had similar elastic constituents but no fiber formation and a
paucity of elastic microfibrils. The subepithelial dense concretions showed strong staining for lysozyme, the first component to be identified in these aggregates. Amyloid P and lysozyme are characteristic components of dermal elastosis, postulated to have an inhibitory effect on elastolytic processes,
indirectly affecting the control of elastogenesis. The greater prominence of nonfiber-forming aggregates in pingueculae may be related to their marked deficiency of elastic microfibrils compared with
dermal elastoses. This difference speaks for more severe actinic cellular damage in the poorly protected
conjunctiyal tissue. Invest Ophthalmol Vis Sci 32:1573-1585,1991
serum protease inhibitors, to elucidate further the
composition of the abnormal elastotic aggregates and
the implied defects in elastogenesis.
The elastic-staining material in pingueculae has
been subject to many interpretations over the years,
particularly when it was discovered that the apparent
elastic fibers were resistant to elastase digestion, leading to the concept that they represented degenerate
collagen.1"3 A more recent ultrastructural study suggested that both newly synthesized elastic fiber precursors and abnormal maturation or dysplasia of elastic fibers formed the largest component of pingueculae.4
Research to date has been able to identify, only to a
limited extent, the abnormal elastotic materials by inference from their similarity to normal elastic structures. Morphologically and ultrastructurally pingueculae have many features in common with cutaneous actinic and breast elastosis5'6 in which immunochemical studies have shown the presence of several
serum components and protease inhibitors.5"9 In the
current electron microscopic study, we used markers
for elastic tissue and related components, including
Materials and Methods
Tissue Preparation
Four conjunctival pingueculae were obtained as
surgical specimens from patients of 50-70 yr of age.
As controls for antibody localization, three bulbar
conjunctival specimens from nonpinguecular areas
were obtained from eyebank eyes in the same age
group. Portions of the tissue werefixedin 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide for
epoxy embedding. Another portion was fixed in 3%
paraformaldehyde, 0.075 M lysine, and 0.01 metaperiodate for 10 min at room temperature, followed
by thorough rinsing in phosphate-buffered saline, gradient dehydration in dimethylformamide, and embedding in Lowicryl K4M (Poly Sciences, Warrington, PA) with ultraviolet light polymerization.
From the Departments of Ophthalmology and Pathology, State
University of New York Health Science Center at Syracuse, Syracuse, New York.
Supported in part by research grant EYO1602 from the National
Eye Institute, National Institutes of Health.
Submitted for publication: September 10, 1990; accepted October 31, 1990.
Reprint requests: Dr. Barbara W. Streeten, Department of Pathology, Weiskotten Hall, SUNY Health Science Center, 766 Irving
Avenue, Syracuse, NY 13210.
Antibodies
Markers for elastic tissue components used in this
study included polyclonal rabbit anti-human aortic
alpha-elastin (Elastin Products, Owensville, MO) and
anti-bovine zonular microfibrillar protein BZ32. The
latter antibody was made to a 32-kD bovine zonular
peptide isolated from a 4 M urea, 50 mM dithiothrei-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1991
tol zonular extract in a polyacrylamide gel band. The
band was used to produce zonular elastic microfibrillar antisera in rabbits as described previously for other
zonular antigens.10 This antibody was made monospecific by affinity purification and showed specific binding to various elastic microfibrils in many other tissues and species. Markers for elastic tissue-associated
components were polyclonal rabbit anti-human amyloid P protein (a gift from Dr. Martha Skinner11),
monoclonal antibody to human vitronectin (Calbiochem, La Jolla, CA), and polyclonal anti-human lysozyme (BioGenex, San Ramon, CA). Markers for
serum protease inhibitors included polyclonal antibodies to human alpha-1 antitrypsin, alpha-2-antichymotrypsin, and alpha-2 macroglobulin (BioGenex). Polyclonal antibodies to other amyloid-related substances, amyloid A protein, human
prealbumin, and human lambda and kappa immunoglobulin (Ig) light chains (Calbiochem), were also
used. All antibodies were monospecific by radioimmunoassay or immunoelectrophoresis.
Immunoelectron Microscopy
The ultrathin sections of both epoxy- and Lowicryl
K4M-embedded tissues were mounted on uncoated
nickel grids. The indirect immunogold procedure was
used for immunostaining. Antielastin antibody was
applied on both epoxy and Lowicryl sections. All
other antibodies were used on Lowicryl sections only.
The grids were incubated in primary antibodies overnight at 4°C, then incubated with colloidal gold-conjugated goat anti-rabbit or goat anti-mouse IgG (EY,
San Mateo, CA) for 30 min at room temperature. Before all incubations, the grids were treated with 3%
nonfat dry milk for blocking nonspecific sites. Between antibody incubations the grids were rinsed
three times for 10 min each with phosphate-buffered
saline. The grids were pretreated with 4 M urea, 50
mM dithiothreitol before reacting with BZ32 antibody and trypsinized before reacting with antibodies
to serum protease inhibitors.6'7 After immunolabeling, Lowicryl sections were submerged in 2% osmium
tetroxide for 10 min, rinsed in distilled water, air
dried, and stained with uranyl acetate and lead citrate.
The specificity of the immunolabeling was tested with
one or more of the following controls: (1) the sections
were processed without primary antibody; (2) the primary antibodies were replaced by antisera adsorbed
with human aortic alpha-elastin, human amyloid P
protein, or lysozyme; or (3) ultrathin sections were
digested by elastase type IV (Sigma, St. Louis, MO)
5mg/ml for 60 min at room temperature12 before immunolabeling with antielastin or antiamyloid P component.
Results
Antibody Localization on Adult Elastic Fibers
in the Conjunctiva
On normal adult elastic fibers, elastin antibody
binding (Table 1) was limited to the electron-lucent
amorphous area of thefibers(Fig. 1 A). The surrounding elastic microfibrils were demonstrated specifically
by microfibrillar antibody BZ 32 (Fig. IB), which did
not bind to the amorphous elastin-containing region,
or to the densities found as aging changes in the center
of adult elastic fibers. Amyloid P component was localized only to the junction between the peripheral
microfibrils and the amorphous elastin (Fig. 1C). Lysozyme was identified scantily at this junction and at
junctions with any remaining microfibrils in the
center of the elasticfiber(Fig. 1D). Virtually no alpha1-antitrypsin or other serum protease inhibitors could
be demonstrated immunologically on normal elastic
fibers.
Antibody Localization on Elastotic Materials
in Pingueculae
As seen by light microscopy of l-/zm epoxy sections, most pingueculae had a thin rim of collagen
Table 1. Antibody reactivity on elastotic materials in pinguecula
Ribbon-like
Non-fiber-forming aggregates
Antibody
Elastin
Microfibrillar
Amyloid P
Vitronectin
Lysozyme
a-1 Antitrypsin
Fibrogranular Polymorphic
Concretions matrix
bodies
fibers
On the
fiber
Thick elastotic fibers
On edge Electron- Electron- On edge
of
lucent
dense
of
Elastic
fiber
areas
inclusions
fiber
microfibrils
0
tr
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Young elastic fibers
++
-+
-+
0
0
+++
0
0
0
+
+++
++
0
+
+
tr
+
tr
0
+++
0
0
0
. 0
Electronlucent
areas
+++
0
0
0
0
0
Junctional
areas
0
0
tr
0
tr
0
No. 5
ELASTIC FIBER COMPONENTS IN PINGUECULA / Li er al
Fig. 1. Gold-labelled antibody localization on adult elastic fibers
in conjunctiva and skin. (A) Elastin antibody labelling the pale
amorphous elastin in a conjunctival elastic fiber (X40.500). (B) Microfibrillar antibody binding to the peripheral elastic microfibrils,
not to the elastin-containing region. Conjunctiva (X40.500). (0
Amyloid P antibody localized on junction of the pale elastin and
peripheral microfibrils. Conjunctiva (X40,500). (D) Lysozyme antibody binding scantily at the junction of elastin and microfibrils,
within the fiber itself. Skin (X40300).
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1575
fiber bundles directly under the epithelium (Grenz
zone8, Fig. 2). In this relatively acellular subepithelial
region, the collagen fibrils ofien showed fraying into
fine filaments, and there were no elastic fibers. Occasional areas of calcification interrupted this area (Fig.
2, zone l), displaying many calcospheres or laminated
areas of linear calcification (Fig. 3A). Subjacent to this
superficial region, sometimes merging with the calcospheres, were angular electron-dense concretions
(Fig. 2, zone 2), between which was a loose fibrogranular matrix (Fig. 3). Lysozyme antibody showed
strong localization on the dense concretions and on
smaller densities in the fibrogranular matrix between
them (Fig. 3B). Elastin was absent in the concretions
and present only on the fibrogranular pale matrix
(Fig. 3C). Localization of amyloid P component was
primarily on the periphery of the concretions and diffusely on the fibrogranular matrix (Fig. 3D), like elastin. Microfibrillar protein antibody was present very
scantily in the fibrogranular matrix but not on the
concretions.
There were a moderate number of collagen fibrils in
this area (Fig. 2, zone 2) between the concretions, composed of 2-3-nm fine helically arranged filaments partially blumng the normal collagen periodicity (Fig. 4).
Also in the same area were tightly aggregated bundles
of 60-70-nm collagen fibrils, with many more fibrils
per unit area than in normal conjunctiva. Collagen
abnormalities were limited to this subepithelial region
of the pinguecula.
In the next deeper zone (Fig. 2, zone 3), which had a
pale homogeneous appearance by light microscopy,
there was a transition from concretions to large polymorphic granular bodies (Figs. 5A-B). Lysozyme antibody showed strong binding to the densest polymorphic areas and was less intense in the more loosely
aggregated regions. Elastin was absent on the densest
materials and their associated matrix, as it was on the
concretions, but was diffusely present on some of the
loose surrounding fibrogranular material (Fig. 5C).
Amyloid P component had a similar diffuse distribution on the loose reticular matrix in some of the polymorphic densities and was absent on the densest regions (Fig. 5D). In the inner parts of this zone, the
polymorphic granular areas were much less electron
dense than the outer and were primarily amyloid P
containing. Collagen was scanty in this zone.
The next deeper area (Fig. 2, zone 4) was a cellular
region with numerous small ribbon-like fragments.
Thin fibroblast cell processes encircled aggregates of
ribbons, partially compartmentalizing them (Fig. 6).
In this heterogeneous zone there were many different
forms of elastic aggregates. On small moderately electrondense ribbons (Fig. 7A), there was co-localization of elastin and amyloid P diffusely on the ribbons.
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Vol. 32
Fig. 2. Pinguecula. Toluidine blue staining (X277).
Zone I. Calcification and fibrosis under the epithelium.
Zone 2. Concretions predominate. Red blood cells
on the left. Zone 3. Polymorphic granular bodies. Zone
4. Region of small, ribbonlike and thick, malformed
elastic fibers.
4
There were also paler ribbons with elastin diffusely
located on them and amyloid P in a more normal
peripheral distribution, along with microfibrillar protein (Fig. 7B), scanty alpha-1 antitrypsin (Fig. 7C),
and lysozyme. In several areas these paler ribbons had
fibrogranular aggregates between them which showed
no elastin staining (Fig. 8A) but a marked accumulation of amyloid P component (Fig. 8B) and small
amounts of microfibrillar protein and vitronectin
(Fig. 8B, inset).
Scattered in this region, but primarily in its deepest
level, were numerous, thick, malformed elastic-like
fibers, replacing the thinner ribbons (Fig. 9). Isolated
whorls of elastic microfibrils were seen (Fig. 10A).
These and some microfibrils on the periphery of the
large elastic-like fibers were labeled by microfibrillar
antibody. Differences in electron density were noted
in these large abnormal fibers. Elastin was present in
the more electron-lucent regions, avoiding the dense
inclusions (Fig. 10A). Microfibrillar antibody localized to the pale areas of these abnormal elastic fibers
and on pale aggregates in which microfibrils could not
be discerned (Fig. 10B). Amyloid P component was
primarily located on the pale areas of these thick
fibers (Fig. 11 A) as it was in the superficial polymorphic granular densities. Some binding also occurred
on the darker areas and the periphery. The more normal the ultrastructural appearance of the fiber, the
more likely it was to have a normal labeling pattern in
comparison with adjacent, more poorly formed
fibers. Lysozyme was again found on the large irregu-
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lar densities in the abnormal elastic fibers (Fig. 11B)
and on the periphery of less well-formed fibers. Alpha- 1 antitrypsin had a scanty diffuse distribution on
the less electron-dense abnormal fibers (Fig. 11C).
The other serum protease inhibitors showed no immunoreactivity on the elastotic aggregates. Collagen fibrils were more normal in this ribbon and fiber-forming elastic region.
Cells in Pingueculae
Fibroblasts, macrophages, mast cells, and rare lymphocytes or plasma cells were found in pingueculae,
mostly in the inner areas. The deeper, better-formed
elastic materials had a very close relationship with fibroblasts, sometimes in contact with the cell membranes of active fibroblasts (Fig. 8). Thin fibroblast
cell processes were interwoven among lobulated elastotic materials, clusters of microfibrils, and densely
aggregated collagen fibrils. Mast cells, both intact and
degranulating, were scattered throughout the lesion,
with free granules often near the elastotic fibers. Cellcell contact between mast cells and fibrobtasts was
seen (Fig. 12A). The mast cell intracellular granules
labeled predominantly with lysozyme antibody (Fig.
12B). Macrophages in the lesion were engulfing elastotic-dense material with some present in their cytoplasm. Interestingly, no elastin antibody was seen on
elastotic material in these cells, although it was present on many of the large densities just outside the
cells.
J
Fig. 3. Angular dense concretions in subepithelial region (zones 1 and 2). Immunogold labelling. (A) Calcospheres lying above the concretions (X8160). (B) Lysozyme antibody localized to the dark irregular concretions (X44,l00). <C) Elastin antibody bound to fibrogranular
matrix, absent on concretions (X44,100). (D) Amyloid P antibody bound to periphery of concretions and on fibrogranular matrix (X44,100).
Fig. 4. Abnormal collagen
fibrils fraying into filaments
in zone 2. Lysozyme antibody labelling of small densities (original magnification X44,IOO).
. • » . ' *
w..
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / April 1991
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Fig. 5. Polymorphic granular bodies in zone 3. (A) Transition of dense concretions to polymorphic granular bodies <x 11,000). (B) Polymorphic bodies in deeper stroma are almost all granular {X11,000). (C) Elastin antibody binds only to looser fibrogranular material around the
polymorphic bodies (X40,500). (D) Amyloid P antibody binds to periphery of densest material and reticular matrix within polymorphic
densities (X40,500).
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ELASTIC FIBER COMPONENTS IN PINGUECULA / Li er ol
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1579
Fig. 6. Cellular zone 4
showing numerous, small
ribbon-like elastic fragments among long fibro- $
blast processes (original
magnification X8300).
Discussion
Identification of some of the elastic matrix components in pinguecula by immunostaining was helpful
in understanding the abnormal forms and in recognizing the progression to more normal aggregation
deeper in the lesion. In the subepithelial region (zones
1 and 2), the concretions and other nonfiber-forming
aggregates gave no ultrastructural evidence of a relationship to elastic tissue, but positive immunologic
evidence (the presence of elastic-associated proteins, a
moderate amount of elastin, and minimal reactivity
for microfibrillar protein). Amyloid P normally localizes on the periphery of elastic fibers after the age of
4-6 yr.13 Its strong reactivity along the edges of the
concretions suggests that these dense structures have
some surface-binding properties similar to those of
normal elastic fibers. The concretions themselves labeled only for lysozyme, a protein noted histochemically on mature elastic fibers in several areas of the
body.7 The collagen fibers in these zones were small
with extensive fibrillar fraying, although not the complete fraying seen by Austin et al.4 A variation in collagen fibril diameter and unraveling of its filaments
have often been attributed to an abnormality of the
proteoglycan matrix,14 although we have no specific
data on this possibility.
The next deeper zone (zone 3), which appeared homogeneous and somewhat eosinophilic by light microscopy, was an interesting transitional zone between the superficial concretions and the innermost
region of very abnormal but definite elastic fiber formations. The confluence of some concretions with
the looser, polymorphic granular densities indicates
that they bear some relationship to each other. Like
the concretions, the polymorphic granular aggregates
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contained no or minimal amounts of elastin, but
amyloid P was the predominant component identified, rather than lysozyme. No other amyloid-related
proteins were detected, and there was minimal microfibrillar protein, A considerable amount of elastin was
present, however, around the polymorphic densities
and on small amorphous fibrogranular clumps. The
fibrogranular matrix and clumps in zones 2 and 3
thus showed all components of the mature elastic
fiber but had not aggregated into fibers, possibly because of their small microfibrillar component
(thought to be the template for elastic fiber formation).
In zone 4, deep to the polymorphic densities, there
was a change to dysmorphic aggregates, recognized by
light microscopy as "elastotic fibers." The marked increase in active and apparently normal fibroblasts in
this region may be important to the ability to produce
fibrous elements. These elements, although abnormal, often appeared arranged linearly beside the long
tapering fibroblast processes as though deposition
were being guided by the cell. The localization of elastin on the small ribbons resembling immature elastic
fibers and in electron-lucent areas in the less wellformed fibrous aggregates was the most significant
sign of their increasing normality. Very few elastic
microfibrils, however, were associated with these fiber
forms although large dysplastic whorls of isolated microfibrils were sometimes adjacent.
The presence of microfibrillar protein on some of
the softer ribbon-like fibers indicated that microfibrillar epitopes were not yet completely hidden by the
binding of amorphous elastin. However, this colocalization is not normal, even for young fibers. In
fact, the presence of normal components that were
out of place was the most characteristic feature of
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / April 1991
Vol. 32
Fig. 7. Ribbon-like elastic fibers in zone 4. (A) Elastin antibody staining of the homogeneous dense ribbons (X67,500). Inset: Amyloid P
antibody has similar localization (X53,1OO). (B) Microfibrillar antibody labellingperiphery of paler ribbons (X51,000). (C) Alpha-1 antitrypsin
antibody scantily labelled on periphery of paler ribbons (X51,000).
these fibrous aggregates. Amyloid P co-localized with
elastin and there was microfibrillar protein over some
of them; in others it localized normally to the periphery. The large dark inclusions dotting electron-lucent
areas were reminiscent of densities in aging elastosis
but contained some aberrant amyloid P component
and the usual lysozyme. The few elastic fibers with a
relatively normal labeling pattern in this region indicated that the factors necessary for normal elastogenesis were present at least focally.
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In analyzing the elastic fiber-like aggregates in pinguecula, it is clear that very few fibers can be considered simply immature. They showed an aberrant organization which is more accurately described by the
words "dysplasia," and in the more superficial formless regions, "dystrophy," as suggested by Austin et
al.4 The component most strikingly deficient was the
basic unit of the elastic fiber, the microfibril. It was
surprising that so much elastin was present without
corresponding microfibrillar protein, but clearly there
ELASTIC FIBER COMPONENTS IN PINGUECULA / Li er ol
No. 5
f
» «• f
•
1581
-*:..
• •
*"• v i > . » ' . '
' t
«
•••
**•
B
Fig. 8. Elastotic aggregates among ribbon-like fibers in zone 4. (A) Elastin antibody labelling dense ribbons, but not fibrogranular aggregates
between ribbons (X67,5OO). (B) Amyloid P antibody labelling periphery of some ribbons but more on the central fibrogranular aggregates
(X67,5OO). Inset: vitronectin antibody scantily bound to the fibrogranular aggregates and periphery of ribbons (X51,000).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1991
Fig, 9. Thick malformed
elastic-like fibers in deeper
stroma among many fibroblast processes (original
magnification X8400).
f
Fig. 10. Thick elastotic fibers in zone 4. (A) Elastin antibody labels electron-lucent area of thick elastotic fibers, avoiding the electron dense
inclusions. Whorls of elastotic microfibrils seen superiorly (X40,500). (B) Microfibrillar antibody labelling electron lucent area, besides
periphery of fibers (X4O,5OO). Inset: labelling of whole abnormal granular elastic fiber by microfibrillar antibody (X40,500).
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ELASTIC FIBER COMPONENTS IN PINGUECULA / Li er al
f
Fig. II. Thick elastotic fibers in zone 4. (A) Amyloid P labels
periphery, as well as central pale and dark areas of abnormal elastotic fibers (X4O,5OO). (B) Lysozyme antibody labels mostly the irregular densities in the elastotic fibers (X4O,5OO). (C) Alpha-1 antitrypsin antibody bound to large, moderately dense fiber (X40?500).
was no fiber formation without microfibrils. It is possible that abnormal microfibrillar protein fragments
were present which lacked epitopes for the 32-kD antibody, but if so these were not fiber forming. Since
there is obvious overproduction of elastic microfibrils
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1583
along with many immature elastic fibers in the usual
dermal actinic elastosis,9 pingueculae may represent
more severe actinic damage to the thinly covered conjunctival cells.
Both normal and abnormal elastic fibers appear to
be produced by the stromal fibroblasts, suggesting a
dearth of fibroblasts in the more severely affected superficial area may correlate with the inability to produce a fibrous elastica. Macrophages and mast cells
have also been noted to be involved in actinic elastosis8 but were not frequent in pinguecula. Cell-cell
contact between mast cells and fibroblasts was seen
occasionally. Such contact was suggested to be a
unique feature of chronic dermal actinic damage, possibly mediating mast cell degranulation by intracellular communication. 15 The protease inhibitors reported in breast elastosis would be expected to derive
from macrophages although mast cell granules contain most of the antiproteases and lysozyme.16 In the
current study, cellular staining for lysozyme and alpha-1 ahtitrypsin, the most common enzymes identified in pingueculae, was most often seen on mast cell
granules, offering support for a mast cell role in this
elastosis. Roles for each of these components will only
be understood by a chronologic study of pingueculae.
The strong positive staining for lysozyme is the first
identification of a component in the concretions.
Electron-dense aggregates of this type in the conjunctiva and cornea have resisted identification, other
than for a proteinaceous composition with evidence
of sulfhydryl and disulfide groups.17'18 By energy dispersive x-ray analysis,19 corneal concretions had consistently high sulfur peaks and some calcium, although calcium was not seen histochemically. Lysozyme is present normally on many elastic fibers and
may be protective against elastolytic enzymes.7'20'21 In
pingueculae it was most consistently present on the
periphery of the ribbon-like elastic fibers but most intensely on the electron-dense concretions and other
dark elastotic inclusions. It has been hypothesized
that lysozyme, a highly cationic protein, binds electrostatically to polyanionic groups such as proteoglycans and glycoproteins, giving it a possible role in carbohydrate remodeling of elastic fibers.22 Lysozyme
has no known substrate for enzymatic activity in
mammalian tissues, but its frequency in connective
tissues implies an important role which may include
the regulation of calcification.20 What lysozyme is
complexed to in the concretions is unknown, but it
could be related to calcium deposition in and adjacent
to them.
Amyloid P component and vitronectin are both
serum proteins which have been localized to the periphery of elastic fibers after 4-6 yr of age.13-23 Amyloid P component is thought to bind to elastic fibers
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INVESTIGATIVE OPHTHALMOLOGY 6 VI5UAL SCIENCE / April 1991
Vol. 32
Fig. 12. Mast cells in zone 4. (A) Mast cell with various stages of degranulated granules showing contact with a fibroblast (X17,0l0). Inset:
high power of contacting area in box (X22,O5O). (B) Lysozyme antibody label on granules in a mast cell (X26,150).
l
by calcium-dependent mechanisms. 24 Vitronectin
was not common in pingueculae, and its role is unknown, but amyloid P has been shown to inhibit elastase activity.23-24 It is not clear whether amyloid P
could be exerting an antielastolytic effect in pinguecula, but it did co-localize with elastin in the coarse
matrix around the concretions and in some of the
immature and poorly formed elastic-like fibers in the
deep zone.
Interest in the possibility that other protease inhibitors might be involved in elastosis, possibly in synergism with amyloid P,24 was raised by evidence of active elastogenesis in these lesions.6 The serum protease inhibitors are not found in normal elastic fibers
or in skin elastosis but have been seen in the immature elastic fibers of breast carcinoma.6*7 In pingueculae only a small amount of alpha-1 antitrypsin could
be demonstrated on the periphery of the small ribbonlike elastotic fibers and scantily on the thick elastotic
fibers. It has been suggested that these inhibitors are
only present on recently formed elastic fibers,7 and it
is likely that there are few young elastic fibers in an
established pinguecula.
Hogan et al2 found that the large elastotic fibers in
pinguecula were elastase insensitive compared with
small presumed normal elastic fibers in the deeper
zone. The regions we found, with large amounts of
amyloid P and lysozyme and some alpha-1 antitrypsin, correlated well with the areas resistant to elastase.
However, when we treated the grids with elastase, the
typical thick elastotic fibers said to be elastase insensitive by light microscopy had no remaining elastin
binding. It is not clear why such fibers would still be
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Verhoeff positive. Either the Verhoeff stain is not specific for the elastin epitope itself, or some of the elastin
is so abnormal it cannot be recognized by the antibody. Alternatively, elastase may be much more effective on ultrathin sections.
It is evident that the control of elastogenesis is seriously defective in pinguecula and that the elastic
fibers are not immature but abnormal in their biochemical organization. The binding of substances
with protease inhibitory action may affect control of
elastin synthesis by reducing elastolysis. In pinguecula, compared with the usual actinic elastosis, there
appears to be a marked reduction of elastic microfibrils rather than overproduction, preventing normal
assembly of elastic fibers.
Key words: pinguecula, elastosis, lysozyme, protease inhibitors, immunoelectron microscopy
References
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2. Hogan MJ and Alvarado J: Pterygium and pinguecula: Electron microscopic study. Arch Ophthalmol 78:174, 1967.
3. Ansari MW, Rahi AHS, and Shukla BR: Pseudoelastic nature
of pterygium. Br J Ophthalmol 54:473, 1970.
4. Austin P, Jakobiec FA, and Iwamoto T: Elastodysplasia and
elastodystrophy as the pathologic basis of ocular pterygia and
pinguecula. Ophthalmology 90:96, 1983.
5. Ledoux-Corbusier M and Danis P: Pinguecula and actinic elastosis: An ultrastructural study. J Cutan Pathol 6:404, 1979.
6. Davies JDand Mera SL: Elastosis in breast carcinoma: H. Association of protease inhibitors with immature elastic fibers, J
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7. Mera SL, Lorell CR, Russell JR, and Davies JD: Elastic fibres
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