a2-Macroglobulin Is Present in and Synthesized by ...

a2-Macroglobulin Is Present in and Synthesized by the Cornea Sally S. Twining,* Takeo Fukuchi,f Beatrice Y. ]. T. Yue,\ Patricia M. Wilson, * Xiaoye Zhou, * and Gerald Loushin*

Purpose. The purposes of this study were to determine whether the proteinase inhibitor a2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. Methods. a2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. Results. a2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the a2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. Conclusions. Because a2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms. Invest Ophthalmol Vis Sci. 1994;35:32263233.

X he maintenance of corneal transparency is highly dependent upon the integrity of corneal proteins, especially the extracellular matrix proteins. Proteinases are present in the cornea and catalyze the normal turnover of proteins.12 Under inflammatory conditions, proteinases are released from polymorphonuclear leukocytes and macrophages as they respond to cor-

From the *Departments of Biochemistry and Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin, and the ^Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois. Supported by grants from the National Institutes of Health, RO1-EY06663 (SST), R01-EY03890 (BYJTY), RO1-EY05628 (BYJTY), and P30-EY01931 (core grant for vision research). Submitted for publication December 9, 1993; revised February 22, 1994; accepted February 24, 1994. Proprietary interest category: N. Reprint requests: Sally S. Twining, PhD, Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.

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neal infection.3 Additionally, bacteria can produce and release proteinases into the cornea.4 The cornea also synthesizes and/or activates proteinases as a response to inflammation and as part of the wound healing mechanism.5 Proteinase inhibitors in the cornea protect corneal proteins against degradation by proteinases under normal and disease conditions. The cornea is known to contain proteinase inhibitors directed toward serine proteinases, a 1-proteinase inhibitor,6 plasminogen activator inhibitor,7 and at least one of the matrix metalloproteinase inhibitors (TIMP).8 Additionally, a 1-proteinase inhibitor9 is synthesized by the cornea. These proteinase inhibitors are single polypeptide competitive inhibitors of single classes of proteinases. In contrast to these inhibitors, a2-macroglobulin is an unusual multifunctional proteinase inhibitor. It

Investigative Ophthalmology & Visual Science, July 1994, Vol. 35, No. 8 Copyright © Association for Research in Vision and Ophthalmology

a2-Macroglobulin in the Cornea

reacts with and inhibits most proteinases from all four major classes: serine, metallo, aspartic, and thiol proteinases.10 This inhibitor is composed of four identical 180-kd subunits10 that form dimers joined by inter-subunit disulfide bridges. These dimers associate into the tetrameric form by noncovalent interactions. a2-Macroglobulin contains a 25-amino acid exposed loop (residues 675 to 700) called the bait region, which is present on each subunit. Proteinases from all classes cleave peptide bonds within this loop. Cleavage rapidly alters the conformation of a2-macroglobulin,n resulting in the physical entrapment of the proteinase within the inhibitor. A thiol ester linkage is also broken, exposing reactive glutamyl and cysteinyl groups on a2-macroglobulin. The glutamyl group reacts with the c-amino group of available lysine residues on either the exposed surface of the proteinase12 or on other proteins in the vicinity. In addition, the active cysteinyl residue can bind peptides or proteins with a free cysteine residue. Up to two proteinase molecules are bound per a2-macroglobulin molecule. Because the proteinases are bound through accessible lysine residues rather than their active sites, captured proteinases are still active toward short polypeptides. Access to the active site of the enzyme is limited by steric hindrance. The purpose of this study was to determine whether the multifunctional proteinase inhibitor, a2-macroglobulin, is present in and synthesized by the cornea. MATERIALS AND METHODS Human Corneas Normal human eyes were obtained either from the Wisconsin Lions Eye Bank (Milwaukee, WI) or from the Illinois Eye Bank (Chicago, IL) within 36 hours of enucleation. The donors had no known ocular diseases. All corneas were examined, and those with opacities and epithelial defects were rejected. Methods for securing human tissue were humane. They included proper consent and approval, and they complied with the tenets of the Declaration of Helsinki. Immunolocalization of a2-Macroglobulin in Corneal Sections Eleven corneas from 2-, 16-, 17-, 19-, 26-, 35-, 40-, 55-, 65-, and 90-year-old donors (35 years ± 26 years, mean ± SD) were processed as previously reported.9 Briefly, corneas were removed with a scleral rim, fixed in 4% formalin, and embedded in paraffin. The corneal sections (5 /im) were deparaffinized, blocked with normal goat serum, reacted with polyclonal rabbit anti-human a2-macroglobulin (Athens Research Technology, Athens, GA, or Dako, Carpenteria, CA, 1:100

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dilution) followed by biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, 1:250 dilution). The sections were then incubated in 0.3% H2O2-methanol to inactivate peroxidases, reacted with avidin-biotin horseradish peroxidase complex (ABC, Vector Laboratories) and developed in 0.01% H2O2, 3,3-diaminobenzidine tetrahydrochloride (Sigma, St Louis, MO). The oxidized substrate was visualized as brown granular products. Control sections were incubated with normal rabbit IgG at a concentration equal to the specific antibody levels. These sections were further processed in the same way as the experimental sections. Corneal Extracts Corneas from donors 18-, 24-, 58-, 66-, 71-, 72-, 78-, 81-, 85, 86-, and 90-years-old (66 years ± 24 years, mean ± SD) were dissected into epithelial, stromal, and endothelial-Descemet's layers and homogenized in 0.1 M Tris buffer, pH 7.6, containing 0.154 M NaCl.6 The homogenates were centrifuged at 13,000g for 10 minutes at 4°C. The supernatant fractions were stored frozen at —80°C until used. Western Blot Analysis for Identification of ct2Macroglobulin Supernatant fractions of the corneal extracts were electrophoresed on 6% SDS-polyacrylamide gels (PAGE) under nonreducing conditions.13 A control sample of human plasma a2-macroglobulin (Athens Research Technology) and cross-linked phosphorylase by molecular weight standards (Sigma) were simultaneously electrophoresed. After electrophoresis, the proteins were electroblotted to nitrocellulose (0.2 /xm, Schleicher and Schuell, Keine, NH). The blots were blocked with nonfat dry milk (Carnation, Los Angeles, CA) in 20 mM Tris buffer, pH 7.6, 0.154 M NaCl, 0.1% Tween-20 (TBS-T), then washed and probed according to the protocol recommended for use with the enhanced chemiluminescence detection system (ECL, Amersham, Arlington Heights, IL). The first antibody used was either a mouse monoclonal antibody (Medix Biotech, Foster City, CA) or a rabbit polyclonal antibody (Athens Research Technology), and the second antibody was either goat anti-mouse IgG antibodies or goat anti-rabbit IgG (BioRad, Hercules, CA) conjugated to horseradish peroxidase. Control blots were incubated with nonspecific mouse IgG (BioRad) or nonspecific rabbit IgG (Sigma) at the same concentration as the specific IgG directed toward a2-macroglobulin. The immunospecific bands were visualized using a luminol-based chemiluminescence ECL detection system (Amersham). Lanes of the blot containing molecular weight standards (Sigma) were stained with india ink (Koh-I-Noor Rapidograph, Bloomsbury, NJ).

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Dot Blot Assay for Quantification of a2Macroglobulin Corneal samples (50 n\) were loaded onto nitrocellulose in a 96-well Dot Blot Apparatus (Schleicher and Schuell). a2-Macroglobulin (Athens Research Technology) was applied at concentrations between 3 and 250 ng to provide a standard curve. The dot blots were probed in the same manner and with the same antibodies used in the Western blots. Nonspecific binding was determined on duplicate blots using nonspecific mouse IgG or rabbit IgG. They were treated as the control Western blots were treated. The labeled dots were quantified using an ELISA plate reader (EL380 Microplate Reader, BioTek Instruments, Winooski, VT) with a 530-nm filter. Each sample was assayed in triplicate. The values for the various samples were averaged, and the standard deviation was determined. Proteins were determined according to the Lowry method14 using bovine serum albumin (Sigma) as the standard. DNA was quantified using the Hoechst 33258 reagent (Sigma)15 with salmon sperm DNA (Sigma) as the standard.

Organ Culture Corneas from donors 23-, 56-, 58-, 60-, 61-, 63-, 64-, 67-, 75-, 75-, 77-, 79-, 81,- and 85-years-old (65 years ± 1 6 years, average ± SD) were dissected with a 2-mm scleral rim from each eye. Each cornea was cultured in 1 ml leucine-, lysine-, and methionine-deficient minimum essential medium (Gibco-BRL, Grand Island, NY) with Earle's salts supplemented with 52 ng L-leucine, 72 /xg L-lysine, 100 /*Ci 35S-methionine (Amersham, cell labeling grade) and 2.2 mg/ml NaHCO3. The organ culture was performed in the presence of 95% air:5% CO2 at 37°C for 24 hours. At the time of harvest, the scleral rims were removed and the corneas dissected into epithelial, stromal, and Descemet's membrane—endothelium. Each corneal layer was frozen in liquid nitrogen, freeze fractured, and extracted in 10 mM Tris buffer, pH 7.2, containing 0.154 M NaCl, 10 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM ethylenediaminetetraacetate (EDTA), 5 fiM E-64, 1 /xM pepstatin, 1% non-idet P-40, 1% sodium deoxycholate, and 0.1% SDS (0.4 ml for endothelium and epithelium and 1.0 ml for stroma or whole cornea). The proteinase inhibitors, PMSF, EDTA, E-64 and pepstatin (Sigma), were used to prevent degradation of a2-macroglobulin during the extraction and assay procedures.

Immunoprecipitation Protein A-agarose (Sigma) was used to immunoprecipitate the a2-macroglobulin-antibody complexes. Before use, the protein A-agarose was hydrated and washed with TxSWB composed of 0.1 M Tris buffer,

pH 8.0, 10 mM EDTA, 0.1 M NaCl, and 1% Triton X-100. The corneal extracts (0.4 ml per endothelium and epithelium, 1.0 ml per stroma) were preabsorbed overnight at 4°C with protein A-agarose (60 mg dry weight) to remove endogenous IgG. The fractions were then absorbed with nonspecific rabbit IgG bound to protein A-agarose. The supernatant fractions were incubated with rabbit anti-a2-macroglobulin and 60 mg dry weight of hydrated protein A-agarose. The resin was centrifuged and washed 5 times with TxSWB. The proteins were eluted by boiling in 2 X PAGE sample buffer containing 25 mM DTT. Samples were electrophoresed on 10% polyacrylamide gels under reducing conditions and stained with Coomassie brilliant blue. The gels were then processed for fluorography using the scintillant, 2,5-diphenyloxazole dithiothreitol, and exposed to x-ray film (Konica, Tokyo, Japan).

In Situ Hybridization of cDNA to a2Macroglobulin mRNA In situ hybridization of a2-macroglobulin mRNA to H-labeled cDNA was carried out using a procedure developed for keratin mRNA in the cornea by Zhu et al.1(> Briefly, nine corneas from donors 16-, 17-, 19-, 22-, 31-, 35-, 40-, and 41-years-old (27.3 years ± 10.6 years, mean ± SD) were fixed immediately in 4% formalin in 0.08 M phosphate buffer, pH 7.0, for 24 hours, processed, and embedded in paraffin. Corneal sections (5 fim) were cut, deparaffinized, rehydrated, and digested with proteinase K (Boehringer Mannheim, Indianapolis, IN). After digestion the sections were washed, postfixed in 4% paraformaldehyde, dehydrated, and dried. Escherichia coli HB101 containing a pKT218 plasmid inserted with 2.1 kB cDNA to a2-macroglobulin (clone pha2ml) was obtained from American Type Culture Collection (Rockville, MD). The cDNA insert contained the coding sequence for the C-terminal portion of the molecule corresponding to amino acids 809 to 1451.17 3H-Labeled DNA probes were prepared using random primers (Amersham) and 3HdATP (ICN, Irvine, CA) according to the method of Haseba et al.18 The mRNA in the corneal sections was hybridized to labeled probe (2 X 106 cpm/ml, 0.1 ^g probe/ml) at 45°C overnight. To test for specificity of binding, additional corneal sections were incubated using the same amount of labeled probe plus 10 ng unlabeled probe/ml under the same incubation conditions as experimental sections. The corneal sections were washed as described9 and then treated with 960 U/ml SI nuclease (Sigma). After rinsing, the slides were dehydrated, air dried, coated with 50% Kodak NTB-2 nuclear emulsion (IBI, New Haven, CT), and exposed for 6 to 8 weeks. The tissue was developed 3


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and counterstained in Mayer's hematoxylin (Sigma) and mounted in Permount (Fisher Scientific, Itasca, IL). RESULTS a2-Macroglobulin was observed in the epithelial, stromal, and endothelial cells (Fig. 1) when corneal sections were immunostained. Epithelial staining was associated with the cytoplasmic area of the cells. The stromal cells stained heavily for this inhibitor. Diffuse staining was seen throughout the extracellular matrix. The inhibitor was also observed associated with the endothelial layer. Neither Bowman's membrane nor Descemet's membrane showed staining for a2-macroglobulin. The immunohistology results were confirmed by Western blot analysis of extracts from the three layers of the cornea using either monoclonal or polyclonal antibodies directed toward a2-macroglobulin. A single band was observed at 360 kd in extracts of stroma, epithelium and Descemet's membrane-endotheiium electrophoresed under nonreducing conditions (Fig. 2). This band comigrated with the dimer form of plasma a2-macroglobulin. Control blots incubated with nonspecific IgG were blank (not shown).

The average total amount of a2-macroglobulin in the stroma per cornea was about two and a half times that of the epithelium (Table 1) as determined by immunodot blot analysis. Endothelial values were obtained by pooling Descemet's membrane-endothelial layers from four different corneas. Extracts of epithelial, stromal, and Descemet'sendothelial layers from corneas metabolically labeled with 35S-methionine were electrophoresed on SDSpolyacrylamide gels under reducing conditions. The viability of the corneal layers was confirmed by the detection of multiple-labeled proteins present in electrophoretically separated extracts of the tissues (data not shown). Only the stromal extracts contained newly synthesized a2-macroglobulin (Fig. 3). This band comigrated with a2-macroglobulin isolated from plasma (not shown); under reducing conditions, it has a molecular weight of 180 kd, which corresponds to the monomer form. In situ hybridization studies showed localization of mRNA in the epithelial, stromal, and endothelial cells (Fig. 4). The background level was low as observed in the control (Fig. 4C) and in the noncellular Bowman's membrane area (Fig. 4B). Silver grains observed in the stroma are mainly associated with the flattened keratocytes (Figs. 4B and 4C). The associa-

B

FIGURE 1. Typical immunolocalization pattern of «2-macroglobulin in a 44-year-otd normal human donor cornea. Paraffin sections of normal human corneas were stained with polyclonal rabbit anti-human a2-macroglobulin (A, C, D, and F) or with nonimmune rabbit IgG (B and E) using an avidin-biotin-peroxidase complex method. Positive reaction products (oxidized diaminobenzidine) appear as granular deposits. Bar = 20 taxi.

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- 3 6 0 kDa - 1 8 0 kDa

O I

o

E o

o E o

CO

CO

Q_ LJ

LU

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in

FIGURE 2. Identification of a2-macroglobulin as a component of the epithelial (Epi), stromal, and Descemet's membrane endothelial (Endo) of human corneas by immunologic identification on Western blots. Corneal extracts were electrophoresed on nonreduced 6% SDS-polyacrylamide gels and then electroblotted to nitrocellulose. Extracts applied to the gel were equivalent to 6% of a cornea (90 jig protein) for the epithelium, 1.25% of the cornea (11 ^g protein) for the stroma, and 24% of the cornea (40 fig protein) for the endothelium. The blots were probed with a mouse monoclonal antibody to «2-macroglobulin. kDa = Kilodalton.

tion of grains with the endothelial cells was striking (Figs. 4A and 4D). Scattered silver grains were also associated with the epithelial cells.

DISCUSSION a2-Macroglobulin was shown to be present in the epithelial, stromal, and endothelial layers of the cornea by immunolocalization and by Western blot analysis of

TABLE l.

o E o

FIGURE 3. Presence of metabolically labeled a2-macroglobulin in stromal extracts of corneas incubated with 35S-methionine in organ culture for 24 hours. Immunoprecipitated protein samples were prepared with 25 mM dithiothreitol and separated on 6% SDS polyacrylamide gels. kDa = Kilodalton.

extracts of the three layers of the cornea. The distribution of this inhibitor is similar to that observed for od-proteinase inhibitor.6 There is, however, a greater relative intensity of staining for a2-macroglobulin in the stromal fibroblasts than that seen with al-proteinase inhibitor. ImmunologicaHy, corneal a2-macroglobulin is the same or similar to that found in the blood. This was demonstrated by the immunorecognition of corneal a2-macroglobulin by several polyclonal and monoclonal antibodies to plasma a2-macroglobulin. The 360kd dimer was detected on the Western blots under nonreducing conditions. This is the form observed for both the tetramer and dimer when separated under nonreducing SDS polyacrylamide gels. There are also

Levels of o:2-Macroglobulin in Normal Human Corneas Hg/Cornea

Epithelium Stroma Endothelium-Descemet's Membrane*

Vg/mg Protein

Vg/*ng DNA

10

7

0.93 ± 0 .08 2.30 ± 1 .31

0.60 ± 0. 03 2.64 ± 1. 04

10 .3± 1.6 34 .9± 17.3

7

0.015 ± 0 .002

0.090 ± 0 . 019

ND

The average age of the corneas used for the determination of a2-macroglobulin in the epithelium was 52 ± 28 years, the stroma was 72 ± 21 years, and the endothelium was 63 ± 10 years. * Values from seven pools of five corneas each were averaged. ND = not determined.

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FIGURE 4. Detection of a2-macroglobulin mRNA in human cornea by in situ hybridization of cDNA probes. Representative corneal sections from a 40-year-old donor were hybridized with 3H-labeled cDNA probes to a2-macroglobulin (total cornea [A] and enlargement of the anterior cornea [B] and the posterior cornea [D]) or labeled cDNA probe mixed with unlabeled probe to serve as a negative control (C). Photos are give as dark-field negatives. Arrows show representative silver grains associated with stromal keratocytes. Arrowheads point to silver grains associated with the endothelial cells. Bars = 20 ^m.

homologues of a2-macroglobulin that possess proteinase inhibitory activity yet have fewer than four subunits.I!) These include pregnancy zone protein (a dimer found in humans) and aj -inhibitor 3 (a monomer in rat and hamster). Whether the immunoreactive c*2-macroglobulin found in the cornea occurs as a tetramer or dimer cannot be discerned from the current studies. The corneal levels of a2-macroglobulin are about 20- to 30-fold less in terms of micrograms per cornea than they are for al-proteinase inhibitor. The molar concentration based on the tetrameric form was 200to 300-fold less. This is greater than the difference between the two inhibitors in serum where the concentration is approximately the same in milligrams per milliliter.20 The corneal concentration of a2-macroglobulin is about 40 times greater that of tears20 and eight times greater that of the aqueous humor.21 Because the experiments reported here were carried out using eye bank donor corneas, there is the potential for the reported levels of a2-macroglobulin to be elevated due to diffusion of this molecule from the limbal blood vessels or other parts of the eye. This, however, does not occur to any great extent. The levels

of a2-macroglobulin present in the epithelial and stromal layers of the eye bank corneas are similar to those determined for keratoplasty corneas removed due to the presence of scar tissue (29.7 fig/mg DNA for the stroma and 10.2 ^g/mg DNA for the epithelium) (Sawaguchi et al, submitted for publication). In the human, the main site of «2-macroglobulin synthesis is the liver. This organ supplies most of this inhibitor found in blood. Extrahepatic tissues that synthesize this inhibitor include the ovary, testis, uterus, placenta, skin and lung fibroblasts, and monocytesmacrophages.10-^ To this list we now add cornea. Newly synthesized 35S-methionine-labeled «2-macroglobulin was found only in stromal layer extracts of organ cultured corneas. This is surprising because of the high level of hybridization between S-methionine-labeled a2-macroglobulin; how-

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ever, by in situ cDNA hybridization, there is a2-macroglobulin mRNA. Immunolocalization and Western blot analysis both show the presence of immunoreactive inhibitor in the epithelial extracts. This difference in the total amount of inhibitor and the apparent amount of synthesis could be due to several factors. In the in situ hybridization experiments, the mRNA may have a short half-life, and much of the mRNA could have been degraded before the tissue was processed. In the organ culture experiment, the culture conditions used may not be ideal for synthesis of this molecule by the epithelial cells. Another possibility is that the epithelial cells do not synthesize much, if any, of the inhibitor. Instead, they may act as scavengers for a2-macroglobulin-proteinase complexes via a receptor-mediated mechanism. Cells that take up a2-macroglobulin through a low density lipoprotein receptorrelated protein (LDLR-RP) include hepatocytes, astrocytes, adipocytes, synctiotrophoblasts, monocytes, fibroblasts, and macrophages. 1023 ' 24 This receptor is distinct from the LDL receptor but is thought to be the ancestral gene of the LDL receptor gene. 10 Whether corneal cells have receptors for a2-macroglobulin-proteinase complexes is not known.

inhibited are proteinases from some bacteria, viruses, and parasites. 10 Because of the size of this molecule (718 kd in the tetramer form) and the avascular nature of the cornea, it is possible that most of the inhibitor present in the stroma is synthesized by the stromal and endothelial cells rather than by diffusing into the cornea from the limbal vessels or from the aqueous humor. In the rat, this inhibitor is an acute-phase protein with synthesis induced by IL-6 via an IL-6 response element. 26 This response element is also found on the human gene, but IL-6 does not induce a2-macroglobulin synthesis in human hepatocytes. However, IL-6 does induce synthesis of a2-macroglobulin in human neuroblastoma cells.27 It is not known whether IL-6 induces synthesis of this inhibitor in the cornea. In summary, a2-macroglobulin is present in the cornea at low levels and is synthesized by corneal cells. This inhibitor is probably a backup to other inhibitors in the cornea, such as a 1-proteinase inhibitor, plasminogen activator inhibitors 1 and 2, and TIMP. It may also be important in the protection and/or clearance of cytokines.

In addition to its role as a proteinase inhibitor in which it reacts directly with proteinases, a2-macroglobulin protects cytokines from degradation 25 by trapping these molecules within its bulky interior. They can also be cross-linked via reactive glutamyl and cysteinyl residues generated during proteinase cleavage of the bait region of a2-macroglobulin. Bound molecules include transforming growth factor-/?, tumor necrosis factor-a, basic fibroblast growth factor, nerve growth factor, platelet-derived growth factor, interleukin-l-/3, and interleukin-6 (IL-6).25 Binding can be mediated via the activated glutamyl and cysteine residues generated upon proteolytic cleavage of the bait region of a2-macroglobulin or by transglutaminases that cross-link proteins to lysine residues on the N-terminal region of the bait region. 10 Most bound cytokines remain active. Some cytokines are more active when bound to a2-macroglobulin than when they are in the free form, whereas others have less activity in the bound form than in the free form. In addition to the protective role of a2-macroglobulin toward cytokines, a2-macroglobulin can also play a role in the clearance of these molecules. 25 The a2-macroglobulin-cytokine complex can bind to and be taken up by the LDL receptor-like protein. It is not known whether a2-macroglobulin-cytokine complexes are found in the cornea. a2-Macroglobulin is probably a key component in the defense of the cornea against proteolytic degradation because it controls the activity of most proteinases. Not only are host proteinases inhibited, but also

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