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

Young elastic fibers

++ -+ -+

0 0 +++

0 0 0

+ +++ ++

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0

+ + tr

+ tr

0 +++ 0 0 0

. 0

Electronlucent areas

+++ 0 0 0 0 0

Junctional areas

0 0 tr

0 tr 0

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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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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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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-

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.

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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).