a?2-Macroglobulin Levels in Normal Human and Keratoconus ... - IOVS

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a?2-Macroglobulin Levels in Normal Human and Keratoconus Corneas Shoichi Sawaguchi* Sally S. Twining,^ Beatrice Y. J. T. Yue* Shirley H. L. Chang,* Xiaoye Zhou,-f Gerald Loushin,\ Joel Sugar,* and Robert S. Feder%

Purpose. To compare the levels of a2-macroglobulin, one of the major proteinase inhibitors, in corneas with keratoconus to those in normal human corneas and corneas with other diseases. Methods. An immunoperoxidase technique was used to visualize the presence of a2-macroglobulin in the corneas. Western blot analysis was performed, and the levels of this inhibitor in extracts of keratoconus and normal human corneas were subsequently analyzed by a dot blot assay. Results. a2-Macroglobulin was demonstrated immunohistochemically in the epithelium, stroma, and endothelium of all corneal sections. Compared with normal human control specimens, the staining intensity in the epithelium of keratoconus corneas was markedly reduced. The majority of scarred and other diseased corneas exhibited normal staining intensity for a2-macroglobulin. Dot blot assays showed that the a2-macroglobulin levels in the epithelial and stromal extracts of keratoconus corneas were lower than those found in normal human control counterparts. Conclusion. Keratoconus corneas contained a reduced level of a2-macroglobulin. This result lends further support to the hypothesis that degradation processes may be aberrant in keratoconus. Invest Ophthalmol Vis Sci. 1994; 35:4008-4014.

l\eratoconus is a progressive disease characterized by thinning and scarring of the central portion of the cornea. 1 ' 2 Biochemical analyses have shown that corneas obtained from patients with keratoconus contain significantly less total protein per milligram of dry weight than those obtained from normal controls.3'4 The protein synthesis in corneal stromal cultures derived from some patients with keratoconus, however, was found to be normal. 3 ' 5 These results have led to the theory that degradation of macromolecules, including proteins, may be one of the mechanisms affected in keratoconus. In support of this hypothesis, we have shown that the levels of lysosomal enzymes, including acid esterase and acid phosphatase, are ele-

From the * Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine; the tDepartments of Biochemistry and Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin; and the %Department of Ophthalmology, Northwestern University, Chicago, Illinois. Supported by research grants EY03890 (BY/r) and EY06663 (SST) and core grant EYOI793 from the National Institutes of Health, Bethesda, Maryland. Submitted for publication fanuary 21, 1994; revised fune 16, 1994; accepted June 23, 1994. Proprietary interest category: N. Reprint requests: Dr. Beatrice Y.J. T. Yue, 1855 West Taylor Street, Chicago, IL 60612.


vated6 and that the levels of an inhibitor, al-proteinase inhibitor, are reduced7 in corneas obtained from patients with keratoconus. The amount of matrix metalloproteinases is unaltered,8 whereas the net activities of gelatinase and collagenase have been found to be greater.9"12 This is possibly because of a decreased level of tissue inhibitor of metalloproteinase9 in keratoconus corneas. We measured the levels of a2-macroglobulin in keratoconus corneas to examine the degradation processes further. This inhibitor and al-proteinase inhibitor are the two major proteinase inhibitors in the plasma.13"15 a2-Macroglobulin, a high-molecularweight (718 kd) homotetrameric glycoprotein, inhibits proteinases from all four major classes.13"15 The mechanism of its action is unique. When a2-macroglobulin reacts with proteinase, proteolytic cleavage in the "bait region" of the inhibitor occurs, leading to a conformational change and trapping of the proteinase.16"18 A covalent bond is then formed between the proteinase and a2-macroglobulin.1516 The proteinase inhibitor complex can be cleared from the circulation by a receptor-mediated mechanism.19'20 or2Investigative Ophthalmology & Visual Science, November 1994, Vol. 35, No. 12 Copyright © Association for Research in Vision and Ophthalmology

a2-Macroglobulin Levels in Keratoconus Corneas Macroglobulin functions as a key defense protein, regulating the degradation of extracellular matrix components and other macromolecules.13 . MATERIALS AND METHODS Fifty normal human eyes from the Illinois Eye Bank, Chicago, and five from the Wisconsin Lions Eye Bank, Milwaukee, were obtained within 24 hours (range, 1 to 24 hours) of death from donors without any known ocular disease. The corneas excised from these eyes were all clear and normal. Thirty-four of the corneas (donor age, 41 ± 21 years; range, 8 weeks to 80 years) were used as controls for immunohistochemical studies, and the remaining 21 (donor age, 53 ± 25 years; range, 14 to 80 years) served as controls for dot blot experiments. Forty-six corneal buttons from patients with clinical features typical of keratoconus were obtained at the time of transplantation. Forty-one corneal buttons were used for immunostaining experiments (patient age, 37 ± 14 years; range, 14 to 87 years; duration of disease, 14 ± 11 years; range, 1 to 41 years), and 16 were used for dot blot assays (patient age, 38 ± 16 years; range, 20 to 87 years; duration of disease, 12 ± 7 years; range, 1 to 27 years). For immunoperoxidase staining,7 normal human and keratoconus corneas were fixed in 10% formalin, processed, and embedded in paraffin. Five-micrometer-thick sections were cut, deparaffinized, rehydrated, incubated first in 10% normal goat blocking serum for 20 minutes and then with polyclonal rabbit antihuman a2-macroglobulin antibodies (1:100 dilution; Dako, Santa Barbara, CA) for 1.5 hours. This affinitypurified antibody preparation was monospecific by immunoelectrophoresis against plasma. After incubation with the primary antibody and rinsing with phosphatebuffered saline, the sections were further incubated with biotinylated goat anti-rabbit IgG (1:500; Vector Laboratories, Burlingame, CA) for 30 minutes, and soaked in 0.3% H2O2-methanol for 20 minutes to block the endogenous peroxidase. They were subsequently incubated with avidin-biotin-horseradishperoxidase complex (ABC; Vector) for 30 minutes, and the color reaction was developed with 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical, St. Louis, MO).7 The sections were dehydrated, mounted in Permont (Fisher Scientific, Itasca, IL), and photographed. The brown color shown in the reaction products was examined under light microscopy. For negative controls, normal rabbit IgG was used in place of the primary antibody. Paraffin sections of corneal buttons from 25 patients (patient age, 70 ± 11 years; range, 50 to 92 years) with pseudophakic (PBK) or aphakic (ABK) bullous keratopathy, eight patients (patient age, 46 ± 21 years; range, 7 to 66 years) with corneal scar, three patients (70, 75, and 79 years of age) with Fuchs' corneal dystrophy, one patient (45

4009 years of age) with lattice corneal dystrophy, one patient (45 years of age) with macular corneal dystrophy, and three patients (40, 50, and 66 years of age) with granular corneal dystrophy were used as an additional set of controls. The keratoconus, normal human, and diseased specimens, as well as their negative controls, were always stained simultaneously under identical conditions, and comparisons were made on all sections stained at the same time in one experiment. Immunostaining experiments were repeated at least three times to confirm the results. In some experiments, cryostat sections from freshly frozen corneas were used. The immunohistochemical staining was analyzed with a Zeiss scanning electron microscope-image processing system (SEM-IPS; Carl Zeiss, Thornwood, NY) established and calibrated21 in Dr. Paul Knepper's laboratory. The measurements were made as percent of transmission with a planapochromatic 40 X oil-immersed objective using a Zeiss scanning microspectrophotometer with the wavelength set at 500 ± 10 nm.6>7>22>23 For each layer of the corneal specimens, measurements on five cells from different fields were made. The measurements were highly reproducible. The transmission values measured were used to calculate the absorbance values by A = log 1/T, where A is the absorbance and T is the percent transmission value. Data obtained from keratoconus and from the central portion of normal human corneas were calibrated against the nonspecific background values obtained with nonimmune normal rabbit serum. Results were analyzed by grouped Student's Mests. For Western blot and dot blot analyses, the central region of normal control corneas was obtained using a 7.5-mm trephine. These pieces, as well as keratoconus and other diseased corneas, were separated into stromal and epithelial layers,7 and each was pulverized in liquid nitrogen and homogenized at 4°C in 150 //I (for epithelial layer) or 500 y\ (for stroma) of 0.1 M Tris buffer, pH 7.2, and 0.154 M NaCl, using a motorized glass homogenizer. Aliquots of the homogenates were taken for measurements of DNA content.24 In some samples, 0.05% hexadecyltrimethylammonium bromide was added to the homogenates. The homogenates were centrifuged at 4°C at 27,000g for 20 minutes. The protein contents in the supernatants were measured by Bradford's protein assay (Bio-Rad, Richmond, CA) using bovine serum albumin as a standard. The amount of protein contained in the pellets was determined after solubilization in 0.2 M NaOH and 0.2% sodium dodecyl sulfate. For Western blot analyses, aliquots of the supernatant fractions (10 fj\) and a2-macroglobulin (Athens Research Technology, Athens, GA) were electrophoresed on 6% sodium dodecyl sulfate gels under reducing conditions. The proteins were electroblotted overnight onto a nitrocellulose membrane (0.2 //m;


Investigative Ophthalmology & Visual Science, November 1994, Vol. 35, No. 12

Schleicher & Schuell, Keine, NH). After blocking with 5% nonfat dry milk (Carnation, Los Angeles, GA), the membrane was allowed to react with either rabbit antia2-macroglobulin (1:10,000; Dako) or rabbit IgG (1:10,000; Cappel, Durham, NC), followed by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000; Cappel). Immunoreactive bands were visualized using the luminol-based enhanced chemiluminescence (ECL) system (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and recorded on x-ray film (Konica, Tokyo, Japan). Prestained molecular weight markers (Bio-Rad) were run in parallel. For dot blot analyses, aliquots (50 //I, in serial dilutions) of each corneal sample were loaded onto a nitrocellulose membrane in a 96-well dot blot apparatus (Bio-Rad) as previously described.7"25 ^-Macroglobulin inhibitor (Athens) isolated from plasma was used as a standard. Immunodetection was carried out as described. The labeled spots visualized by ECL were quantified by a microplate reader (Biotek, Burlington, VT). Data from triplicate samples were averaged. Nonspecific binding was determined on duplicate blots incubated with only normal rabbit IgG and the secondary antibody. These were subtracted from the total binding to determine the specific binding. Resultant values were then compared with the standard curve obtained with known amounts of the inhibitor. The dot blot values were all normalized to the DNA content. Analysis of variance was used to determine the statistical significance of the data. The tenets of the Declaration of Helsinki were followed, and the research was approved by the University of Illinois Institutional Human Experimentation Committee.

RESULTS The diagnosis of keratoconus was confirmed by the results of pathologic examinations. Typical features of keratoconus, including breaks in Bowman's membrane, epithelial edema, thinning in the central cornea, positive iron staining for Fleischer's ring, and mild fibrous scars, were seen. Figure 1 shows the typical immunohistochemical staining patterns of normal human, keratoconus, and other diseased corneas using paraffin sections and a polyclonal antibody specific for a2-macroglobulin. Similar to that shown previously,26 the inhibitor was found in the epithelial, stromal (Fig. 1A), and endothelial (not shown) layers of normal human corneas. In the stromal layer, the inhibitor was observed intracellularly in stromal cells and extracellularly associated with stromal lamellae. The staining pattern and the staining intensity were similar in the normal human specimens, irrespective of donor ages (8 weeks to 80 years). Compared with the controls (Fig. 1A), the staining in keratoconus

corneas (Fig. IB), especially in the epithelial layer, was markedly reduced. Reduced staining was seen in thinned areas as well as in areas seemingly retaining normal thickness. The decrease of the inhibitor was observed in 36 (88%) of the 41 keratoconus specimens examined. Unlike keratoconus corneas, all surgical specimens obtained from patients with lattice and granular corneal dystrophy had normal a2-macroglobulin staining (photographs not shown). Specimens from patients with corneal scars (Fig. 1C) showed strong staining against anti-a2-macroglobulin in the scarred areas. The staining intensity in the nonscarred areas ranged from enhanced (3 out of 8) or normal (4 out of 8) to reduced (1 out of 8). Twenty of the 25 specimens from patients with either PBK (Fig. ID) or ABK, and 2 of the 3 specimens from patients with Fuchs' corneal dystrophy, also had normal staining, whereas the remaining few showed reduced staining. Immunostaining experiments using frozen sections yielded results similar to those using paraffin sections. The immunohistochemical staining was further analyzed, and the intensity of reaction products was measured with the use of an image-processing system. In the central portion of normal human corneas, the stromal cells contained a smaller amount of a2-macroglobulin (Table 1) than did the corneal epithelial and endothelial cells. No differences were found between cells in the anterior and the posterior portions of the stroma. In keratoconus, the corneal epithelium—including basal epithelial, surface epithelial, and wing cells—had a significantly lower level of or2-macroglobulin than that found in normal human corneas. The decrease in the level of the inhibitor was observed in a majority of the keratoconus specimens. For instance, among the nine keratoconus specimens studied, eight had a significantly reduced a2-macroglobulin level in the surface and basal epithelial layers. Six specimens showed such a reduction in the wing cells. The average a2-macroglobulin intensity in the stromal and endothelial cells in corneas with keratoconus was also lower than that in controls, but the differences were not statistically significant. By Western blot analyses, one prominent band with an apparent molecular weight of 180 kd immunoreactive to a2-macroglobulin was found in all the corneal stromal extracts examined (Fig. 2). Minor bands at approximately 138 and 90 kd were also present. Compared with the normal control specimens, the protein bands were fainter, indicating that less a2-macroglobulin was present in the stromal extracts of either keratoconus or PBK corneas. As demonstrated,26 one single protein band corresponding to a2-macroglobulin was observed on Western blot analyses in the epithelial extracts of normal human corneas. Such a band, however, was not discernible in epithelial extracts of keratoconus and PBK samples (data not shown), suggesting that the a2-macroglobulin level in these samples was much lower than that in

a2-Macroglobulin Levels in Keratoconus Corneas


FIGURE 1. Immunostaining of the anterior portions of corneas with a polyclonal a2-macroglobulin antibody. Corneas were from a 47-yearold normal subject (A), a 31-year-old patient with keratoconus (B), a 58-year-old patient with traumatic scar (C), and a 53-year-old patient with pseudophakic bullous keratopathy (D). Positive reaction products appear as brown deposits. Original magnification, X40.

normal human corneas. The detection limit presumably was also affected by the sample size (10 //I) that could be applied to the gels. The amount of a2-macroglobulin in the extracts of normal human, keratoconus, and PBK-ABK corneas was quantified further by dot blot assays (Table 2). Aliquots of up to 50 fA were used for dot blot assays, and reproducible quantification was achieved. For keratoconus samples, reduced inhibitor levels found in the epithelial and stromal layers were consistent with the immunohistochemical findings. The dot blot results showed that the inhibitor content in the PBK and ABK samples was reduced. Their values were

lower even than those from keratoconus samples. The addition of 0.05% hexadecyltrimethylammonium bromide, a detergent, to the extraction buffer resulted in similar values (data not shown). DISCUSSION This study demonstrates, by immunologic and biochemical methods, that a2-macroglobulin is present in the epithelium, stroma, and endothelium of keratoconus and normal human corneas. It is also revealed that the inhibitor level is significantly decreased in corneas from patients with keratoconus. The decrease


Investigative Ophthalmology Be Visual Science, November 1994, Vol. 35, No. 12

l. Staining Intensity of a2Macroglobulin in Corneas From Patients With Keratoconus TABLE

Corneal Cells/Regions

Keratoconus Corneas (n=9)

Surface epithelial cells Wing cells Basal epithelial cells Stromal celts Stromal lamellae Endothelial cells

0.160 0.126 0.114 0.120 0.010 0.312

± ± ± ± ± ±

0.027* (8) 0.027* (6) 0.022f (8) 0.017J (1) 0.002J (0) 0.017J (2)

Normal Human Corneas (n = 5) 0.402 0.345 0.357 0.153 0.026 0.359

± 0.067 ± 0.075 ± 0.055 ± 0.034 ± 0.014 ± 0.022

Data (mean ± SEM) are optical densities obtained by an image processing system as described in Materials and Methods. The specimens analyzed were all from a single experiment, immunostained at the same time under identical conditions. Values in parentheses indicate the number of specimens that showed a value significantly lower than that of normal human controls, * P < 0.006 compared with normal human controls. f P < 0.0004 compared with normal human controls. t Not significantly different from normal human controls.

was observed in a majority of the patients with keratoconus. Under reducing conditions, Western blot analysis indicated the presence of the 180-kd a2-macroglobulin subunit in the corneal stroma. Under nonreducing conditions, the 360-kd dimer form was observed in this study (data not shown) and previous studies.26 It is unknown whether the a2-macroglobulin molecule actually exists as a dimer or a tetramer (718 kd) in the cornea, but both forms of the inhibitor are active.1718 The minor protein bands seen at 138 and 90 kd may represent degradation products or proteinaseinduced fragments,27'28 implicating a functional role of a2-macroglobulin in die cornea. Eleven keratoconus and 11 PBK-ABK specimens were subjected to both immunohistochemical and dot blot analyses. For keratoconus samples, the results from the two studies were consistent; each showed a decreased inhibitor level. Conflicting data, however, were obtained from PBK-ABK samples, in which immunostaining for or2-macroglobulin was mostly normal, and yet by dot blot assays, the inhibitor content was invariably found to be reduced. The discordant results were unexpected, and their basis is elusive. It seems that the decrease in the or2-macroglobulin level was observed only after the extraction procedure. Perhaps because of specific pathologic changes in diseased corneas, the inhibitor is altered structurally, is more aggregated, is crosslinked to or bound with other cellular elements into a complex, or is denatured, and it becomes less accessible for extraction. In the course of this study, it was repeatedly noted that, with the same extraction procedure, the protein

extractable from the PBK-ABK corneas was usually less than that from the normal and keratoconus corneas. The soluble protein constituted an average of 10% of the total protein in the stroma and 14% in the epithelium of PBK-ABK specimens, whereas considerably higher percentages (24% to 28% and 28% to 34%) were found in the normal and keratoconus counterparts. In any case, rationalization of the data must await further investigations. Interestingly, using guanidine hydrochloride for extraction, a similar conflict of data was also observed with al-proteinase inhibitor.7'29 The a2-macroglobulin defect may conceivably affect tissue degradation processes in keratoconus corneas. This rinding is compatible with those of our previous lysosomal enzyme and inhibitor studies,6'7 lending support to the hypothesis that an aberration in degradation processes may be one of the major events in keratoconus. As demonstrated previously,6'7 the biochemical abnormality is manifested in the epithelium of keratoconus corneas, further indicating the role of this corneal layer in the disease. Moreover, a2-macroglobulin is implicated in the protection and regulation of cytokine molecules.10 It has been shown that cytokines, including transforming growth factor-/?,30 platelet-derived growth factor,31 tumor necrosis factor,32 basic fibroblast growth factor, interleukin-1 and interleukin-6, can bind to a2-macroglobulin, and the number of such cytokines keeps increasing. In this context, the decreased a2-macroglobulin level in keratoconus may also affect the cytokine balance in the cornea, cultivating additional biologic consequences. The exact mechanism underlying the a2-macroglobulin defect in keratoconus is unknown. The abnormality may be related to a decrease in biosynthesis by corneal cells or an increased degradation of this inhibitor, or it may reflect a change in the tears or in


3 4


2. Western blot analysis of a2-macroglobulin. The stroma extracts from corneas of a normal subject (lane 1), two patients with keratoconus (lanes 2 and 3), and two patients with pseudophakic bullous keratopathy (lanes 4 and 5) all showed a major protein band of immunoreactivity at 180 kd (arrowhead). Minor bands at 138 kd and 90 kd (arrows) were also observed. FIGURE

«2-Macroglobulin Levels in Keratoconus Corneas


2. a2-MacrogioDulin Levels in Corneas From Patients With Keratoconus


at2-Aiacroglobulin (ng/ftg of DNA) in Epithelium


Keraioconus Normal human controls Pseudophakic/aphakic bullous keratopathy Scar

5.16 ± 1.22* (13)

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