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Histochemical Evaluation of Conjunctival Remodelling in Vernal Keratoconjunctivitis

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Histochemical Evaluation of Conjunctival Remodelling in Vernal Keratoconjunctivitis Eye (2003) 17, 767–771 & 2003 Nature Publishing Group All rights reserved 0950-222X/03 $25.00 www.nature.com/eye 1 2 Immuno- AM Abu El-Asrar , A Meersschaert , LABORATORY STUDY SA Al-Kharashi1, L Missotten2 and K Geboes3 histochemical evaluation of conjunctival remodelling in vernal keratoconjunctivitis Abstract Keywords: vernal keratoconjunctivitis; conjunctiva; allergy; tenascin; laminin; Purpose To study the expression of the fibronectin extracellular matrix (ECM) proteins, tenascin, laminin, and fibronectin in the conjunctiva of patients with active vernal keratoconjunctivitis (VKC). Introduction Methods Conjunctival biopsy specimens were obtained from nine patients with active Vernal keratoconjunctivitis (VKC) is a chronic, VKC and 6 normal control subjects. The seasonally exacerbated bilateral external allergic presence and distribution of tenascin, laminin, ocular inflammation characterized by tarsal and fibronectin were assessed microscopically conjunctival giant papillae and limbal 1Department of with immunohistochemical techniques and thickening and opacification resulting from Ophthalmology a panel of monoclonal and polyclonal hyperplasia of conjunctival connective tissue. College of Medicine antibodies directed against tenascin, laminin, Conjunctival remodelling, characterized by King Saud University Riyadh, Saudi Arabia and fibronectin. extensive deposition of the interstitial proteins Results In normal conjunctiva, weak collagen types I, III, IV, V, and VII in the 2Department of immunoreactivity for tenascin was localized to substantia propria, is a hallmark of the lesions Ophthalmology 1,2 the walls of blood vessels in the upper of VKC. University of Leuven substantia propria. Weak immunoreactivity for Inflammation is characterized by the Leuven, Belgium laminin was located at the epithelial–stromal appearance of many extracellular matrix (ECM) junction and in the walls of blood vessels. components in a spatial and sequential pattern 3Laboratory of Staining for fibronectin was absent. In VKC suggesting that mechanisms functioning during Histochemistry and specimens, intense immunoreactivity for embryonic development may be recapitulated Cytochemistry 3 University of Leuven tenascin was noted in the substantia propria during inflammation. Tenascin is a large Leuven, Belgium associated with the inflammatory infiltrate and hexameric ECM glycoprotein that is found in in the perivascular stroma. Intense embryonic tissues, especially at epithelial– Correspondence: immunoreactivity for laminin around all mesenchymal junctions, and in developing AM Abu El-Asrar stromal vessels and fibrillar immunoreactivity brain tissue, where it probably plays a role in Department of among basal epithelial cells were noted. epithelial–mesenchymal induction and Ophthalmology There was no immunoreactivity for migration. Its expression is restricted in adult King Abdulaziz University Hospital fibronectin. human tissues, and re-expression occurs during Airport Road, PO Box 245 Conclusion Our data indicate increased wound healing, tissue remodelling, and in Riyadh 11411 deposition of tenascin and laminin in the tumour stroma. Tenascin is believed to play Saudi Arabia conjunctiva from patients with active important roles in tissue development, wound Fax: 966-1-477 5724/ VKC. Our findings suggest that tenascin healing, and repair, because it mediates several 4775741 E-mail: abuasrar@ and laminin might play distinct roles cellular activities including cell adhesion and ksu.edu.sa in chronic inflammation seen in antiadhesion, migration, proliferation, and 4,5 VKC. differentiation. Laminin constitutes one of the Received: 3 May 2002 Eye (2003) 17, 767–771. doi:10.1038/ major ECM components of the basement Accepted in revised form: sj.eye.6700453 membrane and is known to have effects on 6 November 2002 Conjunctival remodelling in VKC AM Abu El-Asrar et al 768 diverse cell functions such as cell migration, growth, and (CAPPEL, Turnhout, Belgium) diluted 1 : 50. The slides differentiation.6 Fibronectin is a large-molecular-weight for fibronectin detection were incubated with rabbit anti- adhesive glycoprotein that participates in the regulation human fibronectin antibody (DAKO A/S, Glostrup, of cell proliferation and migration, especially during Denmark) diluted 1 : 750. The sections were then wound healing.7 incubated with the biotinylated secondary antibody and Since conjunctival remodelling is a feature of VKC, we reacted with the avidin–biotinylated peroxidase complex hypothesized that the extracellular matrix proteins (Dakopatts, Copenhagen-Denmark). All incubations tenascin, laminin, and fibronectin are upregulated in the were carried out for 30 min at room temperature, then conjunctiva from patients with active VKC. To test our washed in three changes of phosphate-buffered saline at hypothesis, we used immunohistochemical techniques to pH 7.2 for 15 min. The reaction product was visualized study the expression of tenascin, laminin, and fibronectin by incubation for 10 min in 0.05 M acetate buffer at pH in the conjunctiva from patients with active VKC and 4.9, containing 0.05% 3-amino-9-ethyl-carbazole and from normal individuals. 0.01% hydrogen peroxide, resulting in bright-red immunoreactive sites. The slides were faintly counterstained with Harris haematoxylin. Finally, the Patients and methods sections were rinsed with distilled water and Patients coverslipped with glycerol. Slides used as controls for the staining were treated in an identical manner, except that Nine consecutive patients with active VKC seen at the an irrelevant antibody was used in the first step or the outpatient clinic of King Abdulaziz University Hospital primary antibody was omitted. Positive controls were were included in the study. The patients were six males previously positive-staining tissue, and included 7 and three females. The mean age was 12.6 2.3 years samples from the small intestine from patients with (range: 8–15 years). The symptoms mentioned by all the Crohn’s disease and samples from the liver. patients were itching, redness, photophobia, and tearing. Each patient underwent complete ophthalmic examination and the corneal and conjunctival changes Results were noted and recorded. All patients had the limbal There was no staining in the negative control slides. In form of the disease characterized by broad gelatinous normal conjunctival tissue specimens, weak infiltrates of the limbus. Nasal or temporal limbal immunoreactivity for tenascin was limited to the walls of conjunctival biopsy specimens were obtained from each blood vessels in the upper substantia propria (Figure 1). patient. None of the patients was on topical therapy Weak immunoreactivity for laminin was observed at the before obtaining the biopsy. In addition, six limbal epithelial–stromal junction and in the walls of blood conjunctival biopsy specimens were obtained from the vessels (Figure 2). There was no immunoreactivity for same areas in patients undergoing strabismus surgery fibronectin. without obvious inflammation and served as controls. All VKC specimens showed similar findings. Intense The controls were from the same age group. band-like imunoreactivity for tenascin was noted in the upper substantia propria (Figure 3). There was a close association between tenascin expression and the Immunohistochemical staining inflammatory infiltrate (Figure 4). The composition of the The conjunctival biopsy specimens were immediately inflammatory infiltrate in VKC was described in snap frozen in Tissue-Tek optimum cutting temperature previous reports.8–10 In addition, intense (OCT) compound (Miles Laboratories, IN, USA) and immunoreactivity for tenascin was noted in the maintained at À80oC until use. For perivascular areas in the substantia propria (Figure 3). immunohistochemistry, 5 mm serially cut cryostat Intense immunoreactivity for laminin was noted around sections were dried overnight at room temperature, fixed all stromal vessels. In addition, fibrillar in absolute acetone for 10 min, then treated with 2% immunoreactivity for laminin was noted among the basal hydrogen peroxide in methanol for 3 min to block epithelial cells (Figure 5). There was no endogenous peroxidase activity, and stained with a immunoreactivity for fibronectin. three-step avidin–biotinylated peroxidase-labelled complex procedure. The slides for tenascin detection Discussion were incubated with monoclonal mouse anti-human tenascin antibody (DAKO-tenascin, TN2, Carpinteria, In normal conjunctiva, tenascin was weakly expressed in CA, USA) diluted 1 : 50. The slides for laminin detection the walls of blood vessels. This observation was were incubated with polyclonal antilaminin antibody consistent with the findings of previous reports.11 In Eye Conjunctival remodelling in VKC AM Abu El-Asrar et al 769 Figure 1 Immunohistochemical staining for tenascin of con- Figure 3 VKC. Immunohistochemical staining for tenascin junctiva from a normal control subject showing weak immuno- showing intense immunoreactivity in upper substantia propria reactivity in the walls of blood vessels in the upper substantia and around stromal vessels (Â40). propria (Â40). Figure 2 Immunohistochemical staining for laminin of con- junctiva from a normal control subject showing weak immuno- reactivity at the epithelial–stromal junction and in the walls of blood vessels (Â40). contrast, the conjunctiva from patients with active VKC showed increased tenascin immunoreactivity. Our results point to a clear association between the inflammatory Figure 4 VKC. Immunohistochemical staining
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