British Journal of Ophthalmology 1997;81:677–682 677 ORIGINAL ARTICLES—Laboratory science Br J Ophthalmol: first published as 10.1136/bjo.81.8.677 on 1 August 1997. Downloaded from Tenascin-C expression in normal, inflamed, and scarred human corneas H Maseruka, R E Bonshek, A B Tullo Abstract myotendinous associated extracellular matrix Aims/background—In adult tissues the (GMEM) protein,3 myotendinous antigen,4 expression of tenascin-cytotactin (TN-C), hexabranchion,5 and J1-200/220.6 Currently, an extracellular matrix glycoprotein, is this glycoprotein is referred to as tenascin- limited to tumours and regions of con- cytotactin (TN-C).7 tinuous renewal. It is also transiently Several in vitro studies have shown that expressed in cutaneous and corneal TN-C mediates a number of cellular activities. wound healing. There are limited data For example, it stimulates89and inhibits910the regarding its expression in inflammation proliferation of cells. It also interacts with and scarring of the adult human cornea. fibronectin (FN), and supports adhesion of a In this study, TN-C expression patterns in variety of cell types including fibroblasts, glia, normal, inflamed, and scarred human neurons, and endothelial cells.11–13 Paradoxi- corneas have been examined. cally, antiadhesive properties have also been Methods—Penetrating keratoplasty speci- described,12 13 providing evidence for multi- mens were selected from cases of herpes functionality of this glycoprotein. simplex keratitis, herpes zoster ophthal- TN-C shows a time and tissue specific micus, rheumatoid arthritis ulceration, expression in the developing embryo. It is, for bacterial keratitis, rosacea keratitis, inter- example, present at the epithelial mesenchyme stitial keratitis, and previous surgery so as interfaces of skin, oral mucosa, lung, genitouri- to encompass varying degrees of active nary tract, gastrointestinal tract, kidney, breast, http://bjo.bmj.com/ and chronic inflammation and scarring. and in the developing brain and cornea.281415 TN-C in these and in normal corneas was In adult tissues TN-C expression is limited to immunodetected using TN2, a monoclonal tumours, regions of continuous renewal, and antibody to human TN-C. wound healing (reviewed by Sakakura and Results—There was no TN2 immunoposi- Kusano16). tivity in normal corneas except at the Although TN-C is expressed in developing corneoscleral interface. In pathological 15 human fetal cornea, in normal adult human on October 2, 2021 by guest. Protected copyright. corneas, TN2 immunopositivity was local- cornea very low expression has been demon- ised in and around regions of active strated in corneal epithelial cells only,17 with a inflammation, fibrosis, and neovasculari- Department of greater degree of expression at the limbal sation. TN2 positivity was less in acute Pathological Sciences, region.17 18 In rabbits, however, TN-C has been University of inflammation than in active chronic in- detected in the entire normal corneal epithe- Manchester flammation. Mature, avascular scar tissue lium, but it is absent in the stroma.19–21 As seen H Maseruka and epithelial downgrowth were TN2 R E Bonshek in cutaneous wound healing,22 23 TN-C is tran- negative. siently expressed in corneal wound —These results indicate that in Department of Conclusion healing.17 19–21 However, to date there are Ophthalmology, the adult human cornea, TN-C expression limited data regarding the expression of TN-C Manchester Royal Eye is induced in regions of inflammation, in both normal and pathological adult human Hospital fibrosis, and neovascularisation, but that H Maseruka corneas. This, together with a body of evidence expression is absent in mature, avascular R E Bonshek supporting a role for this glycoprotein in influ- A B Tullo scar tissue. This suggests a role for this 2 6 8–13 15 glycoprotein in inflammation, healing, encing several cellular activities, Correspondence to: and extracellular matrix reorganisation of prompted us to examine TN-C expression in Dr Richard E Bonshek, the cornea. inflamed, vascularised, and scarred human Department of corneas. Comparison of patterns of TN-C Ophthalmology, University (Br J Ophthalmol 1997;81:677–682) of Manchester, Royal Eye localisation in pathological and normal corneas Hospital, Oxford Road, was aimed at determining whether a relation Manchester M13 9WH. Tenascin is a large extracellular matrix glyco- exists between the progression of events in ∼ 1 Accepted for publication protein ( 190–320 kD) which has been inflammation, healing, and scarring, and 10 April 1997 described variously as cytotactin,2 glioma- TN-C expression in the human cornea. 678 Maseruka, Bonshek, Tullo Table 1 Source of reagents and antibodies employed ated spirit (IMS). Endogenous peroxidase activity was quenched by a 30 minute incuba- Br J Ophthalmol: first published as 10.1136/bjo.81.8.677 on 1 August 1997. Downloaded from Reagents/antibodies Source tion with 1% (v/v) hydrogen peroxide (H2O2), Mouse anti-human and antigenic sites unmasked by incubation tenascin-C (clone TN2, with 1 mg/ml trypsin in 4 mM CaCl2 and 200 IgG1, kappa) Dako, UK ° Mouse anti-human desmin mM TRIS, pH 7.6 (10 minutes, 37 C). A (clone D33, IgG1, kappa) Dako, UK solution of 20% (v/v) normal rabbit serum Rabbit anti-mouse IgG (NRS) was applied to sections (15 minutes, (RAM) Dako, UK ° Avidin-biotin/horseradish 25 C) to block non-specific binding sites. The peroxidase complex NRS was then tipped oV and sections incu- (ABC/HRP) Dako, UK bated overnight (4°C) with a 1:25 (v:v) TN2. Industrial methylated spirit (IMS) Genta Medical, UK Bound TN2 was detected by applying 1:100 3-Aminopropyltriethoxysilane Sigma Chemical Company, (v:v) biotinylated rabbit anti-mouse immu- (APES) USA noglobulins (30 minutes, 25°C). This was then Trypsin tablets Sigma Chemical Company, USA visualised by incubating sections (30 minutes, Normal rabbit serum (NRS) Sigma Chemical Company, 25°C) with a solution of avidin-biotin horse- USA radish peroxidase complex (ABC/HRP) and Hydrogen peroxide (H O ) BDH Laboratory Supplies, UK 2 2 reacting with a 3,3’diaminobenzidine (DAB) tetrachloride/H2O2 substrate. Sections were Materials and methods counterstained with Harris’s haematoxylin, de- Thirty five corneal specimens obtained from hydrated in graded (70%–99% (v/v)) IMS and patients aged from 3 to 97 years, who had cleared in xylene before mounting coverslips undergone penetrating keratoplasty, were se- with xylene anti-mouse IgG (XAM). Sections lected to encompass varying degrees of acute were viewed under a routine light microscope. and chronic inflammation, and scarring. These Between incubation steps, sections were dip included the following cases—bacterial kerati- immersion washed (2 × 3 minutes) in 0.05 M tis (three); herpes simplex keratitis (HSK) TRIS buVered saline, pH 7.6, to eliminate (10); rosacea keratitis (three); herpes zoster excess non-bound antibody or reagent. Nega- ophthalmicus (HZO) (five); interstitial kerati- tive controls included substitution of TN2 with tis (IK) (seven); rheumatoid arthritis (RA) an irrelevant antibody (mouse anti-human ulceration (five); and previous surgery (two). desmin, clone D33, isotype IgG1, kappa), and Ten globes enucleated for choroidal melanoma with 25% (v/v) NRS. No immunoreaction was but without anterior segment pathology pro- observed in negative controls. Skin from a case vided normal corneas. All tissues were fixed in of chronic dermatitis and sclera provided TN2 10% (v/v) phosphate buVered formalin, pH positive immunoreaction controls. 7.4, before being dehydrated and embedded in paraYn wax. Results Reagents and antibodies employed in the TN2 immunoreaction was negative in all the study are shown in Table 1. A mouse normal corneas except at the corneoscleral anti-human TN-C (clone TN2, isotype IgG1, interface (Fig 1), where sclera was positive. http://bjo.bmj.com/ kappa) obtained from Dako, UK, was em- Generally, in all the TN2 positive corneas, ployed in this study. TN2 has been employed stromal staining was seen mostly in an in a number of studies investigating tissue dis- extracellular location. In cases of bacterial tribution of TN-C.18 24 keratitis, TN2 was strongly positive in corneal Microtome sections (6 µm), mounted on stroma around the periphery of areas of acute 3-aminopropyl-triethoxy silane (APES) coated inflammation and areas of active ulceration (Figs 2 and 3). In areas away from the main slides, were dewaxed in xylene and rehydrated on October 2, 2021 by guest. Protected copyright. in graded (99%–70% (v/v)) industrial methyl- focus of inflammatory activity and where there was separation of epithelium from stroma, there was increased subepithelial staining (Fig 2). At higher magnification (Fig 3) focal TN2 staining was seen in occasional keratocytes/ fibroblasts in acutely inflamed tissue. It was apparent that much of the intense stromal TN2 immunoreaction around the inflamed areas was associated with chronic inflamma- tory cell infiltrate, although the infiltrating cells were themselves negative. An intermittent line of staining was also seen at the junction between Descemet’s membrane and the post- erior stroma (Fig 3). Cases of HSK, rosacea keratitis, and HZO showed heterogeneity of stromal staining for TN2. However, in all cases there was stromal TN2 immunoreactivity associated with active chronic inflammation (Fig 4). As in the chronic inflammation around the area of acute inflam- matory activity shown in Figures 2 and 3, the Figure 1 Normal cornea and corneoscleral interface. There is TN2 immunopositivity of the sclera (small arrows) including the scleral spur (large arrow). Corneal tissue is TN2 inflammatory cells were TN2 negative, and immunonegative (× 18). there was focal positivity of the stroma/ Tenascin-C expression in normal, inflamed, and scarred human corneas 679 In corneas with previous surgery there was TN2 immunostaining in regions of active scar Br J Ophthalmol: first published as 10.1136/bjo.81.8.677 on 1 August 1997. Downloaded from tissue, associated with some chronic inflamma- tory cells, where stromal and lymphocyte cyto- plasmic membrane staining was seen, and some vascular endothelial cells, where there was intracytoplasmic staining (Fig 6).