Gelatinase B in Vernal Keratoconjunctivitis

LABORATORY SCIENCES Gelatinase B in Vernal Keratoconjunctivitis

Ahmed M. Abu El-Asrar, MD, PhD; Ilse Van Aelst, BLT; Samir Al-Mansouri, MD; Luc Missotten, MD, PhD; Ghislain Opdenakker, MD, PhD; Karel Geboes, MD, PhD

Objectives: To investigate the expression of gelatinase thelial and stromal inflammatory infiltrate. Compared with B in the conjunctiva of patients with vernal keratocon- control specimens, VKC specimens showed signifi- junctivitis (VKC) and the cellular source of this en- cantly more gelatinase B–positive cells (mean±SD, zyme. 40.8±29.9 vs 10.3±2.4; PϽ.02). Most gelatinase B–positive cells were eosinophils (90.2%±3.6%). Zymography Methods: Conjunctival biopsy specimens from 12 pa- revealed that gelatinase B levels in VKC specimens were tients with active VKC and 12 control subjects were stud- significantly higher than the levels found in normal con- ied using immunohistochemical techniques and a mono- junctiva (3780.3±3541.0 vs 610.1±397.1 scanning units; clonal antibody against gelatinase B. The phenotype of PϽ.03). gelatinase B+ inflammatory cells was examined using double immunohistochemical analysis and monoclonal Conclusions: These findings suggest overexpression antibodies against eosinophil peroxidase or macro- of gelatinase B by eosinophils in VKC specimens and phage CD68. Quantitative zymography was used to com- participation of gelatinase B in the pathologic changes pare the activity of gelatinase B in conjunctival biopsy in VKC. specimens from 10 patients with active VKC and 7 control subjects. Clinical Relevance: Control of the release and/or activation of gelatinase B in eosinophils may provide a new Results: Gelatinase B was detected in a few polymor- therapeutic strategy for treating VKC. phonuclear cells in 8 control specimens. All VKC specimens showed gelatinase B immunoreactivity in the epi- Arch Ophthalmol. 2001;119:1505-1511

ERNAL keratoconjunctivitis The matrix metalloproteinases are rec- (VKC) is an allergic chronic ognized as key enzymes for normal extra- seasonally exacerbated bi- cellular matrix turnover and for the exag- lateral external ocular in- gerated extracellular matrix breakdown flammation that affects chil- associated with pathologic conditions, in- Vdren and young adults, with a predominance cluding tumor cell invasion and metasta- in males. The disease is characterized by resis, angiogenesis, inflammatory reactions, current symptoms of severe itching, pho- wound healing, and scar formation.10-12 The tophobia, lacrimation, and discharge. There major members of this family include the are 3 forms of the disease: palpebral, lim- following: collagenases, which degrade and bal, and mixed. The classic sign of palpe- denature fibrillar collagen types I, II, and III; From the Department of Ophthalmology, College bral VKC is the giant papillae or cobble- gelatinases A and B (respectively, the 65-kd of Medicine, King Saud stones in the upper tarsal conjunctiva. The to 75-kd matrix metalloproteinase-2 and the University, Riyadh, Saudi limbal form is characterized by gelatinous 85-kd to 96-kd matrix metalloproteinase- Arabia (Drs Abu El-Asrar and infiltrates of the limbus. Corneal findings 9), which cleave denatured collagens (gela- Al-Mansouri); and the are common and include punctate epithe- tins), collagen types IV, V, VII, and X, elas- Laboratory of Molecular lial keratitis, epithelial erosions, corneal tin, and fibronectin; and stromelysins, which Immunology, Rega Institute for ulcers, and plaque formation.1,2 degrade proteoglycans, laminin, fibronec- Medical Research (Ms Van The typical histological features of tin, type IV collagen, and the globular do- Aelst and Dr Opdenakker), the VKC are the conjunctival infiltration by eo- mains of other extracellular matrix macro- Department of Ophthalmology sinophils, basophils, mast cells, B lympho- molecules.10 More recently, membrane- (Dr Missotten), and the + Laboratory of Histochemistry cytes, plasma cells, CD4 T lymphocytes type matrix metalloproteinase expressed on and Cytochemistry expressingT-helper2(TH2)–typecytokines, cell membranes is identified as a fourth cat- 13 (Dr Geboes), University and monocytes/macrophages, extracellu- egory. Hospital St Rafael, University lar matrix hyperplasia, and remodeling Because of its unique and broad sub- of Leuven, Leuven, Belgium. caused by increased collagen deposition.3-9 strate specificity and its involvement in

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 PATIENTS AND METHODS immunoglobulin and the avidin/biotin peroxidase– labeled complex, respectively (Dakopatts A/S, Copenha- gen, Denmark). All incubations were carried out for 30 min- IMMUNOHISTOCHEMICAL ANALYSIS utes at room temperature, then washed in 3 changes of phosphate-buffered isotonic sodium chloride solution at Twelve patients with active VKC seen at the outpatient clinic a pH of 7.2 for 15 minutes. The reaction product was vi- of King Abdulaziz University Hospital, Riyadh, Saudi Ara- sualized by incubation for 10 minutes in 0.05M acetate buffer bia, were included in the study. All the patients were males at pH 4.9, containing 0.05% 3-amino-9-ethyl-carbazole aged 7 to 17 years, with a mean age of 12 years. The symp- (Sigma-Aldrich, Bornem, Belgium) and 0.01% hydrogen per- toms mentioned by all the patients were itching, redness, oxide, resulting in bright red immunoreactive sites. The photophobia, and tearing. Each patient underwent com- slides were faintly counterstained with Harris hematoxy- plete ophthalmic examination, and the corneal and con- lin. Finally, the sections were rinsed with distilled water junctival changes were noted and recorded. All patients had and coverslipped with glycerol. Control slides were treated the limbal form of the disease characterized by broad ge- in an identical manner, except that an irrelevant IgG mouse latinous infiltrates of the limbus. Nasal or temporal limbal monoclonal antibody was used in the first step, or the pri- conjunctival biopsy specimens were obtained from each pa- mary antibody was omitted. tient. None of the patients was receiving topical or sys- temic therapy before obtaining the biopsy. In addition, 12 Double Immunohistochemical Analysis limbal conjunctival biopsy specimens were obtained from the same areas from patients undergoing cataract extrac- To examine the phenotype of gelatinase B–expressing in- tion or strabismus surgery without obvious inflammation flammatory cells, cryostat sections were studied by double and served as controls. The controls were from the same immunohistochemical analysis. Colocalization studies were age group. This study was approved by the Research Cen- performed in 4 VKC specimens, using mouse antihuman ter, College of Medicine, King Saud University (Riyadh, phenotype monoclonal antibodies CD68 (1:1000, macro- Saudi Arabia), and the patients admitted to the study gave phages) (Dakopatts A/S) or eosinophil peroxidase (Ab-1) their informed consent. (1:1000, eosinophils) (Oncogene Research Products, Cam- The conjunctival biopsy specimens were immedi- bridge, Mass) together with REGA-2D9 monoclonal anti- ately snap-frozen in optimum cutting temperature com- body. After rinsing the slides with phosphate-buffered iso- pound (Tissue-Tek; Miles Laboratories, Elkhart, Ind) and tonic sodium chloride solution, they were incubated for 30 maintained at −80°C until use. For immunohistochemical minutes with REGA-2D9 monoclonal antibody and rinsed analysis, 5-µm serially cut cryostat sections were dried over- again with phosphate-buffered isotonic sodium chloride so- night at room temperature, fixed in absolute acetone for lution. Subsequently, the sections were incubated for 30 10 minutes, and stained with a 3-step avidin/biotin per- minutes with peroxidase (EnVision+ system, mouse; DAKO oxidase–labeled complex procedure. Rehydrated slides were Corporation, Carpinteria, Calif) and washed again with incubated for 30 minutes with gelatinase B–specific REGA- phosphate-buffered isotonic sodium chloride solution, and 2D9 monoclonal antibody (1:50). The mouse monoclonal the reaction product was visualized by incubation for 10 antibody REGA-2D9 was raised against natural human neu- minutes in 0.05 M acetate buffer at pH 4.9, containing 0.05% trophil gelatinase B. This implies that the antigen prepa- 3-amino-9-ethylcarbazole (Sigma-Aldrich) and 0.01% hy- ration was devoid of gelatinase A. The REGA-2D9 is an im- drogen peroxide, resulting in red immunoreactive stain- munoglobulin (Ig)G1 subtype with a dissociation constant ing. Afterward, the sections were rinsed in phosphate- (Kd) value of 9.5ϫ10 −10 M, which implies extremely high buffered isotonic sodium chloride solution, washed with specificity.15 This monoclonal was compared with other an- distilled water, and incubated for 30 minutes with the sec- tibody preparations and found to be superior for immu- ond monoclonal antibody to determine cellular pheno- nohistochemical analysis.16 The secondary and tertiary re- type (CD68 or eosinophil peroxidase). After a wash with agents consisted of biotin-conjugated rabbit antimouse phosphate-buffered isotonic sodium chloride solution, the

other chronic inflammatory diseases,14 we hypothesized few polymorphonuclear cells located in the vascular that excessive expression of gelatinase B may play a role lumens and in the perivascular areas in 8 of 12 speci- in extracellular matrix remodeling in VKC. To evaluate mens (Figure 1). All VKC specimens showed immu- this hypothesis, we examined conjunctival specimens noreactivity with the monoclonal antibody against obtained from patients with VKC using immunohisto- gelatinase B in the epithelial and stromal inflammatory chemical analysis and gelatin zymography. The findings infiltrate (Figure 2). In the stroma most of the posi- in VKC were compared with the findings in the con- tively stained cells were located just beneath the epithe- junctiva from normal individuals. lium. The numbers of gelatinase B–positive cells in VKC specimens were significantly greater than the RESULTS numbers found in control specimens (40.8±29.9 vs 10.3±2.4; PϽ.02). IMMUNOLOCALIZATION OF GELATINASE B Double immunohistochemical analysis to confirm the phenotype of gelatinase B–positive inflammatory cells There was no staining in the negative control slides. In showed that most inflammatory cells expressing gelati- normal conjunctiva, gelatinase B was detected only in a nase B were eosinophils (90.2%±3.6%, n=4) (Figure 3).

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 sections were incubated for 30 minutes with a biotin- specimen an equivalent amount of 100 µg protein was ana- labeled rabbit antimouse antibody, followed by a mono- lyzed as described by Masure et al.17 Phosphorylase b (97 clonal anti–biotin-alkaline phosphatase conjugate (Sigma- kd) was used as a standard protein. The localization of ge- Aldrich). The blue reaction product was developed using latinolytic enzymes and their molecular masses were de- 4-benzoylamino-2,5-diethoxybenzene-diazonium chlo- rived by including protein-sizing standards on each gel and ride (Fast Blue BB salt; Sigma-Aldrich) for 5 minutes. on the basis of the known migration of gelatinase B vari- ants (monomer, dimer, and heterodimer with neutrophil Quantitation gelatinase B–associated lipocalin) that were purified by Ma- sure et al.17 Briefly, samples were examined by electropho- Cells were counted in 5 representative microscopic fields. resis in 7.5% polyacrylamide gels that had been copoly- Counting was performed by 2 independent observers merized with 0.1% (weight/volume [wt/vol]) gelatin (A.M.A. and K.G.). One of them (K.G.) was unaware of the (Sigma-Aldrich). Stacking gels contained 5% polyacryl- origin of the specimens. In case of disagreement, the re- amide. Electrophoresis was at 4°C for 16 hours at 90 V. The sults obtained by the blinded observer were used. We used gels were then incubated in washing buffer (50mM Tris- an eye piece calibrated grid (original magnification ϫ25). hydrochloride [Sigma-Aldrich], pH 7.5, 10 mM calcium With this magnification and calibration, we counted the chloride, 0.02% [w/v] sodium azide, 2.5% [v/v] Triton cells present in an area of 0.155ϫ0.155 mm. For the co- x-100) at room temperature, twice for 20 minutes, to re- localization studies, cells expressing both gelatinase B and move sodium dodecyl sulfate and overnight in developing eosinophil peroxidase or CD68 were counted and ex- buffer (50mM Tris-hydrochloride, pH 7.5, 10mM calcium pressed as a percentage of cells expressing gelatinase B. chloride, 0.02% [wt/vol] sodium azide, 1% [vol/vol] Tri- ton x-100) at 37°C. The gels were then stained in Coo- ZYMOGRAPHY massie brilliant blue R-250 and destained in methanol/ acetic acid. The sites of gelatinase activity appeared as Ten male patients aged 7 to 15 years (mean age, 9.5 years) unstained bands on a blue background and were quanti- with severe active VKC were included in the study. All pa- fied by densitometry and zymolytic activities that were ex- tients had the palpebral form of the disease characterized pressed as arbitrary laboratory scanning units. Densitom- by the presence of giant polygonal flat-topped cobble- etry was with a densitometric scanner (PDI, New York, NY), stone papillae affecting the upper palpebral conjunctiva. and the raw data were processed with Pharmacia Biotech Upper palpebral conjunctival biopsy specimens were ob- software programs (LabScan Utility [version 2.00] and Im- tained from each patient after obtaining informed con- age Master ID [version 2.0]; Pharmacia Biotech, Uppsala, sent. None of the patients was receiving topical or sys- Sweden). The linear range was between 200 and 7000 scan- temic therapy before obtaining the biopsy. In addition, 7 ning units. Quantitation of gelatinase activity by zymog- upper palpebral conjunctival biopsy specimens were ob- raphy proved to be a sensitive nonisotopic detection method tained from patients who served as controls and were in a with a sensitivity in the picogram range.18 similar age group undergoing strabismus surgery without obvious inflammation. STATISTICAL ANALYSIS The conjunctival biopsy specimens were immedi- ately snap-frozen in optimum cutting temperature com- All data are presented as mean±SD. The Mann-Whitney pound (Tissue-Tek; Miles Laboratories) and maintained at U test was used to compare the mean numbers of gelati- −80°C until use. For gelatin zymography, frozen tissues were nase B–expressing inflammatory cells in VKC patients with thawed and transferred into 50 µL of phosphate-buffered controls. The t test was used to compare the mean gelati- isotonic sodium chloride solution at a pH of 7.4, supple- nase A and B levels in VKC patients vs controls. Loga- mented with 1% Triton x-100 (Sigma-Aldrich). This prepa- rithm transformation was used to reduce variances before ration was sonicated at 0°C for 3 minutes, and protein con- applying the t test. The differences were considered sig- centrations were determined on a fraction. For each nificant at PϽ.05.

Smaller numbers of inflammatory cells expressing gelati- Gelatinase B values in VKC specimens were signifi- nase B were monocytes/macrophages (10.9%±5.0%, n=4) cantly higher than the values found in control speci- (Figure 4). mens (control specimens, 610.1±397.1 scanning units; VKC specimens, 3780.3±3541.0 scanning units; ZYMOGRAPHY PϽ.03).

Constitutive gelatinase A was detected in 6 of 7 control COMMENT specimens and in 6 of 10 VKC specimens (Figure 5). Gelatinase A values in VKC specimens were higher than Zymography and immunohistochemical analysis indi- the values found in control specimens, but the differ- cated increased activity and expression of gelatinase B ence between the 2 groups was not statistically signifi- in the conjunctiva from patients with VKC compared with cant (control specimens, 210.5±114.1 scanning units; control subjects. The up-regulation of gelatinase B in this VKC specimens, 663.3±484.2 scanning units; P=.08). study is consistent with previous studies that docu- Inducible gelatinase B was detected in all control mented the increased expression of gelatinase B messen- specimens and in 8 of 10 VKC specimens (Figure 5). ger RNA and protein in bronchial biopsy specimens from

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Figure 1. Immunohistochemical staining of conjunctiva from a normal Figure 2. Vernal keratoconjunctivitis. Immunohistochemical staining showing control subject showing immunoreactivity in few polymorphonuclear cells immunoreactivity in the epithelial (arrows) and stromal (arrowheads) (arrow) (gelatinase B, original magnification ϫ500). inflammatory infiltrate (gelatinase B, original magnification ϫ300).

subjects with asthma.19,20 Furthermore, gelatinase B lev- duction remains at the same level after macrophage stimu- els were increased in the bronchoalveolar lavage flu- lation. In contrast, unstimulated macrophages do not ids21-23 and in the sputum24 of patients with asthma. The produce gelatinase B but secrete large amounts of this numbers of eosinophils were correlated with the degree enzyme after appropriate stimulation by cytokines and of expression of gelatinase B in bronchial biopsy speci- endotoxin, for example.12,14,18,27 mens,20 and gelatinase B activity strongly correlated with Double immunohistochemical analysis showed that the numbers of eosinophils and neutrophils recovered gelatinase B immunoreactivity was predominantly asso- in the bronchoalveolar lavage fluid.22 In the present study, ciated with eosinophils present in the inflammatory in- gelatinase A levels did not vary significantly among con- filtrate in VKC specimens. Our observations are consis- junctival biopsy specimens from control subjects and those tent with a previous report that most cells expressing from subjects with VKC. Similarly, levels of gelatinase gelatinase B messenger RNA in bronchial biopsy speci- A levels were low or undetectable in bronchoalveolar la- mens from subjects with asthma were eosinophils.19 Pre- vage fluids from patients with asthma.23 In general, these vious studies indicate that circulating eosinophils28 and observations agree with those of Paemen et al25 for con- eosinophils in normal bronchial tissues19 do not express stitutive gelatinase A distribution in cerebrospinal fluid gelatinase B messenger RNA. Therefore, it is likely that of patients with a variety of short- and long-term inflam- eosinophils are triggered to synthesize gelatinase B in the matory nervous system disorders. Gelatinase A was ex- conjunctiva from patients with VKC. Factors regulating pressed equally in the cerebrospinal fluid of patients and the expression of gelatinase B by eosinophils in VKC are control subjects. Human gelatinase A occurs constitu- incompletely understood; however, certain cytokines may tively in body fluids (serum, synovial fluid, cerebrospi- be involved. The TH2-derived cytokines interleukin-3 (IL- nal fluid) and cell culture supernatants.25 It is suggested 3), IL-5, and granulocyte-macrophage colony- that gelatinase A seems to be involved in some basal ex- stimulating factor prolong eosinophil survival as well as tracellular matrix turnover events, whereas gelatinase B activate these cells.29-32 Recently, Okada et al33 showed seems to be involved in more acutely regulated events.26 that IL-5, platelet-activating factor, or both increased re- In normal human and mouse macrophages and human lease of gelatinase B by eosinophils in vitro. There is strong macrophage cell lines, gelatinase A is constitutively pro- evidence that these TH2-type cytokines are centrally induced in small but detectable quantities, and this pro- volved in the pathogenesis of VKC34 and other allergic

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©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 Figure 3. Vernal keratoconjunctivitis. Double immunohistochemical staining Figure 4. Vernal keratoconjunctivitis. Double immunohistochemical staining for gelatinase B (red) and eosinophil peroxidase (blue) showing gelatinase for gelatinase B (red) and CD68 (blue) showing gelatinase B–positive cells B–positive cells coexpressing eosinophil peroxidase (arrows) (original coexpressing CD68 marker (arrow) (original magnification ϫ1200). magnification ϫ500).

diseases.35,36 In addition, CC chemokines have been rec- hyperresponsiveness in a murine model of allergic asthma. ognized to play an important role in eosinophil recruit- Increased expression of gelatinase B in VKC could also ment and activation.37 Recently, we have shown in- render basement membranes vulnerable and increase vas- creased expression of the CC chemokines RANTES cular permeability, facilitating conjunctival edema and (regulated on activation, normal T cell expressed and se- the transmigration of inflammatory cells through the base- creted), eotaxin, and monocyte chemotactic protein-3 in ment membrane. Indeed, the conjunctiva from patients the conjunctiva from patients with VKC.38 with VKC is characterized by remarkable inflammation The recruitment of eosinophils from the circula- from the aspects of cellular infiltration and the expres- tion to the site of inflammation is regarded as a key event sion of adhesion molecules, as indicated by our previ- in the development and maintenance of allergic inflam- ous studies.3-5 mation. The molecular events involved in the infiltra- Eosinophils are now recognized to play a central role tion of eosinophils through endothelium and the epithe- in the pathophysiologic characteristics of VKC. Strong lium have been investigated intensively in in vivo and basic cytotoxic proteins such as major basic protein, eo- in vitro systems, showing the involvement of adhesion sinophil cationic protein, eosinophil peroxidase, and eo- molecules,39 cytokines,40 and chemokines.35,37 Since one sinophil-derived neurotoxin are released from eosino- of the main components of endothelial and epithelial base- phils and damage the conjunctival and corneal ment membrane is type IV collagen, which is specifi- epithelium.44,45 More recently, eosinophils were recog- cally cleaved by gelatinase B,41,42 eosinophil gelatinase B nized as a source of proinflammatory cytokines, which might permit the extravasation of eosinophils through may act to perpetuate the local immune response.46 In the basement membrane zone underlying endothelial and addition, gelatinase B released from eosinophils may con- epithelial cell layers. Recently, gelatinase B was re- tribute to progressive breakdown of conjunctiva in VKC. ported in an in vitro system to play a crucial role in the Gelatinase B degrades denatured collagens (gelatin), col- transmigration of eosinophils through basement mem- lagen types IV, V, VII, and X, elastin, and fibronec- brane components.33 Furthermore, Kumagai et al43 dem- tin.41,42 This tissue destruction may be followed by re- onstrated that inhibitors of matrix metalloproteinases pre- modeling of the conjunctiva with increased deposition vent the cellular infiltration and the induction of airway of collagens. In a previous immunohistochemical study,

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Control Gel 1 ST 1234567 V6 V7 V8 V9 C1 ST

Complex Complex MMP-9 97 kd 97 kd MMP-9 MMP-2

MMP-2

Gel 2 V1 V2V3 V4 V5 ST VKC ST 1234567 8 9 10

Complex Complex MMP-9 97 kd

97 kd MMP-2 MMP-9

MMP-2 Gel 3 ST C2 C3 C4 C5 C6 C7 V10

Complex 97 kd MMP-9

MMP-2

Figure 5. A, Gelatin zymography of conjunctival biopsy specimens from normal control subjects and from patients with active vernal keratoconjunctivitis (VKC). The zymographies of these samples show the presence of both gelatinase A (MMP-2) and gelatinase B (MMP-9). The standard protein (ST) shows a 97 kd band. (The figure is a montage of several gels.) B, Gelatin zymographies (3 gels) of conjunctival biopsy specimens from normal control subjects (lanes indicated with C) and from patients with active VKC (lanes indicated with V). The zymographies of these samples show the presence of both gelatinase A (MMP-2) and gelatinase B (MMB-9). A protein standard showing a 97-kd band (lanes indicated with ST) was run on each gel. The lanes without indication show different kinds of laboratory samples, including samples with known amounts of gelatinases.

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