Jpn J Ophthalmol 43, 251–256 (1999)© 1999 Japanese Ophthalmological Society 0021-5155/99/$–see front matterPublished by Elsevier Science Inc. PII S0021-5155(99)00021-0

LABORATORY INVESTIGATIONS

TGF-

b

2, Tenascin, and Integrin

b

1Expression in Superior Limbic Keratoconjunctivitis

Akira Matsuda, Yoshitsugu Tagawa and Hidehiko Matsuda

Department of Ophthalmology,Hokkaido University School of Medicine, Sapporo, Japan

Purpose:

To determine the pathophysiological etiology of superior limbic keratoconjunctivi-tis (SLK), we compared the superior limbic conjunctivae of SLK patients and normal con-trols.

Methods:

Frozen sections of conjunctival specimens from five SLK patients and two controlswere examined by immunohistochemical methods. Transforming growth factor (TGF)-

b

2,integrin

b

1, and tenascin (TN) were chosen for analysis because their expression is known tobe affected by mechanical stress or injury. The staining pattern was observed by confocal la-ser scanning microscopy.

Results:

Prominent positive TGF-

b

2 staining on the surface region and heterogeneous stain-ing in the suprabasal region were observed in the SLK specimens. TN expression was mark-edly up-regulated in the subepithelial stroma. In addition, suprabasal expression of integrin

b

1 was observed.

Conclusions:

Up-regulation of TGF-

b

2 and TN suggested that an increased amount of me-chanical stress existed in the conjunctivae of the SLK patients. In addition, deposition of TNand suprabasal expression of integrin

b

1 suggested that chronic minor injury contributed tothe pathogenesis of SLK.

Jpn J Ophthalmol 1999;43:251–256

© 1999 Japanese Ophthal-mological Society

Key Words:

Superior limbic keratoconjunctivitis, transforming growth factor-

b

, tenascin,

integrin

b

1, mechanical stress.

Introduction

Superior limbic keratoconjunctivitis (SLK) is anocular surface disorder of unknown etiology origi-

nally reported by Theodore.

1

Clinically, it occursmost commonly between 20 and 60 years of age; andat least 75% of the cases occur in women, without ra-cial predilection.

2

In a previous study, we reportedthat the limbal conjunctivae of SLK patients had al-terations of cytokeratin expression and showed signsof hyperproliferation.

3

Although the pathogenesis of SLK remains un-known, the mechanical stress between the upper tar-sal and bulbar conjunctiva is believed to be involvedin the pathophysiology of the disease.

4

This hypothe-

sis is supported by the finding that snake-like chrom-atins, which are commonly observed in SLK,

3

are anindication of mechanical stress on the ocular sur-face.

5

On the other hand, the clinical observationthat wearing a hard contact lens can induce SLK

6

suggested that chronic minor injury of the superiorlimbal epithelium might have a possible role in thepathogenesis of SLK.

Mechanical stress can induce the expression ofvarious cytokines as well as the extracellular matrix,which can then alter the differentiation status of epi-thelial cells. Transforming growth factor (TGF)-

b

isa multipotential cytokine that is known to be in-duced by mechanical stress

7

and is expressed in theocular surface epithelium.

8

Mechanical stress canalso induce extracellular matrix tenascin (TN) whoseexpression is controlled spatiotemporally.

9

It was also reported that both TN and integrin

b

1are expressed during corneal wound healing.

10,11

To

Received: March 26, 1998Correspondence and reprint requests to: Akira MATSUDA,

MD, Department of Ophthalmology, Hokkaido UniversitySchool of Medicine, Kita 15 Nishi 7, Sapporo 060-8638, Japan

252

Jpn J OphthalmolVol 43: 251–256, 1999

determine the possible involvement of mechanicalstress and the wound healing process in the patho-genesis of SLK, we examined the expression ofTGF-

b

, TN, and integrin

b

1 in the conjunctivae ofSLK patients.

Materials and Methods

Patients

The superior limbic conjunctivae of five patientswith SLK were resected therapeutically as describedpreviously.

3

Three cases had a severe form of SLKwith hyperemia in the upper bulbar conjunctivashowing intense Rose-Bengal and fluorescein stain-ing, and ridge formation of the superior limbus. Theremaining two patients had moderate cases of SLKwith mild infection and focal Rose-Bengal and fluo-rescein staining in the superior limbic conjunctiva.All SLK patients were Japanese women aged 49, 54,58, 62, and 66 years. Two normal superior limbic con-junctivae obtained during malignant orbital tumorsurgery were also examined. The patients with orbitaltumors were also Japanese women and their ageswere 62 and 64 years. All these specimens were col-lected after obtaining appropriate informed consent.

Immunohistochemistry

Immunohistochemical study was performed essen-tially as described previously.

3

In brief, the speci-mens were immediately embedded in OCT com-pound (Miles, Elkhart, IN, USA), frozen in liquidnitrogen, and preserved at

2

75

8

C until used. Frozensections of 5

m

m were mounted on poly-L-lysine–

coated slides and air dried for 30 minutes at roomtemperature. The slides were fixed in 4% paraform-aldehyde in 0.1 mmol/L phosphate buffer (pH 7.4).After being washed in 0.01 mmol/L phosphate-buff-ered saline (PBS) (pH 7.4), slides were incubated inPBS containing 10% normal goat serum and 1% bo-vine serum albumin for 1 hour at room temperatureto block nonspecific staining. The slides were thenincubated overnight at 4

8

C with the primary antibod-ies listed in Table 1. After washing in 0.05% Tween20 in PBS (TPBS), they were incubated with fluores-cein isothiocyanate (FITC)-conjugated secondaryantibodies. All slides were incubated for 1 hour atroom temperature followed by three washings inTPBS. The staining was observed and recorded witha confocal laser scanning microscope (CLSM), (MC-1024; Bio-Rad Laboratories, Hercules, CA, USA).For negative control, nonimmune rat, mouse, or rab-bit IgG was used instead of primary antibodies at thesame concentrations.

Results

The clinical and laboratory findings in the SLKpatients are summarized in Table 2. Histopathologi-cal analysis of the specimens was the same as pub-lished previously.

3

The fixed sections were stainedeither with hematoxylin-eosin (H-E) (Figure 1) or byindirect immunofluorescence staining with anti–TGF-

b

2, integrin

b

1, and TN antibodies. All sectionswere processed together with the negative controls.The negative control sections did not show any spe-cific staining. The immunohistochemical findings aresummarized in Table 3.

Table 1

. Panel of Primary Antibodies

Antibodies Type/Class Antigen Source

TGF-

b

2 Rabbit polyclonal/affinity-purified IgG Synthetic peptide* corresponding to aminoacid 352–377 of human TGF-

b

2Santa Cruz Biotechnology

(Santa Cruz, CA, USA)Tenascin Rat monoclonal/affinity-purified IgG Purified human tenascin Gift from Dr. M. Kusakabe

20

Integrin

b

1 Mouse monoclonal/purified IgG1 Integrin

b

1 from human fibroblasts Biohit

*Sequence of TGF-

b

2 peptide: YLWSSDTQHSRVLSLYNTINPEASAS

Table 2.

Clinical Findings in SLK Patients

Patients Age Tasal Conjunctiva Ridge Formation Schrimer Test Thyroid Function

A 49 Papillary proliferation Normal HyperthyroidismB 54 Papillary proliferation

1

Normal NormalC 58 Papillary proliferation

1

R 11.5 mm, L 8.5 mm NormalD 62 Papillary proliferation Normal NormalE 66 Papillary proliferation

1

R 9 mm, L 14 mm Normal

A. MATSUDA ET AL.

253

TGF-

b

, TENASCIN, INTEGRIN EXPRESSION IN SLK

Anti–TGF-

b

2 Staining

Anti–TGF-

b

2 staining of normal conjunctivaeshowed staining predominantly in the cytoplasm ofthe basal cells (Figure 2A). The staining pattern ofSLK samples with the anti–TGF-

b

2 antibody wasdifferent from that of the controls. The basal and su-perficial cells showed strong cytoplasmic staining butthe intensity of staining of the suprabasal cells variedfrom cell to cell. Some of the cells that did not showpositive staining were observed as dark spots (Figure2B, 2C). The section reacted with nonimmune rabbitIgG did not show any specific staining (Figure 2D).

Anti-TN Staining

Only faint linear anti-TN staining was observed atthe epithelial–stromal junction and in the perivascu-lar stroma in the normal conjunctiva (Figure 3A). Incontrast, there was intense subepithelial staining ofthe stroma in the SLK sections (Figure 3B).

Anti-Integrin

b

1 Staining

The sections from normal conjunctivae showedpositive peripheral cytoplasmic staining in the basal

cells with the anti-integrin

b

1 antibody (Figure 4A).In contrast, the conjunctivae from SLK patientsshowed positive integrin

b

1 staining in almost theentire epithelium (Figure 4B). Integrin

b

1 was alsopresent in the connective tissue as described previ-ously.

12

Discussion

We have shown an up-regulation of TGF-

b

2 ex-pression in the surface region, and heterogeneousTGF-

b

2 expression as well as ectopic integrin

b

1 ex-pression in the suprabasal region of the specimensfrom SLK patients. In addition, the expression of TNon the subepithelial stroma was prominently up-reg-ulated in the limbal conjunctivae of these patients.

The dominant subtype of TGF-

b

expressed in thelimbal epithelium was reported to be TGF-

b

2.

8

Ourresults are consistent with this in that TGF-

b

2 wasthe predominant subtype found on the limbal epithe-lium. We have also analyzed TGF-

b

1 and

b

3 expres-sion in a preliminary study, and no definitive stainingwas observed (data not shown). Although the TGF-

b

2 antibody used was raised against synthetic poly-

Table 3.

Summary of Immunohistological Results

TGF-

b

2 Tenascin Integrin

b

1

Patients Basal/Suprabasal/Superficial Epithelium/Subepithelium Basal/Suprabasal/Superficial

A

11

/H/

11 2

/

11 11

/

11

/

2

B

11

/H/

11 2

/

111 11

/

11

/

1

C

11

/H/

11 2

/

111 111

/

11

/

1

D

11

/H/

1 2

/

11 11

/

11

/

12

E

11

/H/

11 2

/

111 111

/

11

/

1

Control 1

11

/

1

/

1 2

/

1 1

/

12

/

2

Control 2

11

/

1

/

1 2

/

12 11

/

12

/

2

111

: intense staining;

11

: moderate staining;

1

: weak staining;

12

: faint staining; H: heteroge-neous staining.

Figure 1. H-E staining of conjunctivae from control and SLK patients. (A) Normal superior limbic conjunctiva. Conjuncti-vae from patients with moderate forms (B) and severe forms (C) of SLK. Bar 5 100 mm.

254 Jpn J OphthalmolVol 43: 251–256, 1999

peptides of mature TGF-b2, it is not known whetherit is specific for the active form of TGF-b. Thus thedata presented here should be accepted with thiscaution.

A previous study showed that mechanical stresscould induce TGF-b expression.7 Our study demon-strates prominent TGF-b2 expression, especially onthe surface region of conjunctiva from SLK patients(Figure 1B, 1C). Because the primary mechanicalstress on the SLK conjunctivae was from friction be-tween limbal and tarsal conjunctiva, we believed thisexpression pattern was reasonable. The prominentpapillary proliferation observed in the upper tarsalconjunctivae of all SLK patients supports this hy-pothesis (Table 2). On the other hand, we cannotfully explain the nature of the heterogeneous expres-sion of TGF-b2 on the suprabasal cells in the con-junctivae of the patients. It should be noted thatsome of the suprabasal cells were completely devoidof TGF-b2 staining. This region correlated with theproliferating cell’s nuclear antigen-positive regionfound in our previous study.3 These results suggestedthe coexistence of a heterogeneous cell population atvarious differentiation/proliferation stages in the su-prabasal cells of SLK patients. In addition, the ex-

pression of TGF-b2 in the basal cells did not differsignificantly between normal and SLK conjunctivae(Figure 1). This finding suggested that another factor(eg, serum) may control the basal expression ofTGF-b2. Factors other than mechanical stress thatcould affect the expression/activation of TGF-bshould also be considered. More specifically, vitaminA (retinol), which is known to affect the activationof TGF-b13 may also play a role in the pathogenesisof SLK.14

Another finding in this study was the prominentdeposition of extracellular matrix TN on the subepi-thelial stroma of the conjunctivae of SLK patients(Figure 2B). To our knowledge, this is the first re-port showing an abnormality of the subepithelial tis-sue in SLK. TN is a large hexameric extracellularmatrix glycoprotein that is known to be expressed intumors and during wound healing.9,10 The precise bi-ological activities of TN remain unknown, but it af-fects cell adhesion15 and corneal development.16 Wehypothesized that increased mechanical stress in theconjunctivae of SLK patients can induce prominentTN accumulation on the conjunctival stroma. It iswell known that mechanical stress can induce TN ex-pression.9 As a consequence, increased TN on the

Figure 2. Anti–TGF-b2 staining of limbalconjunctiva. (A) Normal superior limbicconjunctiva. Strong basal staining and rela-tively homogeneous cytoplasmic staining ofsuprabasal cells. (B, C) Conjunctivae fromSLK patients. Intense cytoplasmic stainingin basal and superficial cells and heteroge-neous cytoplasmic staining in suprabasalcells. (D) Section adjacent to figure B wasreacted with nonimmune rabbit IgG. Thisnegative control section does not show anyspecific staining. Bar 5 100 mm.

A. MATSUDA ET AL. 255TGF-b, TENASCIN, INTEGRIN EXPRESSION IN SLK

stroma may in turn alter the differentiation status ofconjunctival epithelial cells and thus contribute tothe pathogenesis of SLK. Other factors may contrib-ute to the TN depositions on the subepithelialstroma of SLK patients. Cytokines or growth factors(eg, interleukin-1, basic fibroblast growth factor, andTGF-b) are known to induce TN expression.17 Thecontribution of these factors to the pathogenesis ofSLK should be examined further.

Suprabasal expression of integrin b1 during cor-neal wound healing has been reported.11 When wecompared the pathological state of SLK and the typ-ical wound healing process, many similarities wereobserved. First, infiltration of inflammatory cells is acommon feature of both conditions. Second, forma-tion of a temporary extracellular matrix such as TNis commonly observed. Third, the local synthesis ofcytokines such as TGF-b, which regulates the forma-tion of granular tissue, is also observed. Consideringthese findings together with the suprabasal expres-sion of integrin b1 (Figure 3B), we suppose that achronic wound healing–like process contributes tothe pathogenesis of SLK.

In summary, we have shown the expression pat-terns of TGF-b, TN, and integrin b1 in the conjuncti-

vae of patients with SLK, and the results suggestedthat mechanical stress contributed to the pathophys-iology of SLK. Although it should be noted thatother factors (eg, cytokines, vitamin A deficiency)might affect the expression patterns, our findingssupport previous evidence for the contribution ofmechanical stress to the pathophysiology of SLK.Taken together with the results of our previousstudy that showed the altered cytoskeleton and pro-liferative status of SLK, we believe SLK to be an ex-cellent pathological model for “tensegrity.” Tenseg-rity hypothesizes that mechanical stress can betransmitted to the cytoskeleton through the cell sur-face receptor integrin and extracellular matrix, andthus could affect cellular architecture and its re-sponse.18 Further studies are needed to clarify therole of mechanical stress on the pathogenesis of SLK.

Recently, the effectiveness of superior lacrimalduct punctal occlusion as a therapy for SLK was re-ported.19 This therapy seems reasonable to improvethe wet ocular surface condition and therefore de-crease mechanical strain and prevent the chronic in-jury–like phenomenon in SLK. We believe that addi-tional therapies for SLK focusing on mechanicalstrain are quite promising.

Figure 3. Anti-TN staining of limbal con-junctiva. (A) Normal superior limbic con-junctiva. Positive anti-TN staining on sub-epithelial stroma as well as perivascularstroma. (B) Conjunctiva from SLK patient.Intense anti-TN staining on subepithelialstroma. Longitudinal section of perivascu-lar stroma (arrow) also showed positiveanti-TN staining. ep: conjunctival epithe-lium; s: conjunctival stroma. Bar 5 100 mm.

Figure 4. Anti-integrin b1 staining oflimbal conjunctiva. (A) Normal superiorlimbic conjunctiva. Positive staining onsurface of basal cells. ep: conjunctival epi-thelium; s: stroma. (B) Conjunctiva fromSLK patient. Note positive integrin b1staining in all layers of limbal conjunctiva.Bar 5 100 mm.

256 Jpn J OphthalmolVol 43: 251–256, 1999

This research was supported in part by a grant-in-aid from theMinistry of Education, Science, Sports and Culture of Japan. AkiraMatsuda is a Research Fellow of the Japan Society for the Promo-tion of Science. We are grateful to Dr. M. Kusakabe for providingthe monoclonal anti-tenascin antibody.

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