Collagen content and AHMED M. ABU EL-ASRAR, KAREL GEBOES, types in trachomatous SOLIMAN A. AL-KHARASHI, KHALID F. TABBARA, LUC MISSOTTEN conjunctivitis Abstract chronic progressive conjunctival cicatrisation in trachoma that lead to blindness remain unclear. Purpose To study alterations in conjunctival However, previous immunohistochemical collagen in the conjunctiva of patients with studies demonstrated that the tissue damage active trachoma. might result from immunological Methods We studied conjunctival biopsy 3 s mechanisms. - specimens obtained from nine subjects with The metabolic alterations of extracellular active trachoma and from four control matrix components in trachoma may be the subjects. We used immunohistochemical result of cytokines released by resident techniques and a panel of monoclonal and conjunctival cells and by numerous polyclonal antibodies directed against types I, 6 inflammatory cell types infiltrating the tissue. III, IV and V collagen. The banded collagen fibrils are the most Results In normal conjunctiva, the staining for abundant and widely distributed matrix types I and III collagen was localised to the components. Increased collagen content is a substantia propria. Type IV collagen was feature of many inflammatory and located in the epithelial and capillary fibroproliferative diseases?-17 endothelial basement membranes. The We analysed conjunctival biopsy specimens staining for type V collagen was absent. In from patients with active trachoma in order to trachoma biopsy specimens, staining for types determine some of the mechanisms that control I and III collagen showed collagen fibrils conjunctival scarring. Using among epithelial cells, patchy increase in immunohistochemical methods we examined staining intensity in the upper stroma, and the ature and distribution of types I, III, IV thicker and irregularly arranged collagen n and A.M. Abu EI-Asrar fibrils in the substantia propria. Staining for V collagen in conjunctival biopsy specimens SA AI-Kharashi with active K.F. Tabbara type IV collagen showed irregularly thickened from patients trachoma. Department of epithelial basement membrane. Staining for qphthalmology type V collagen showed patchy staining in the College of Medicine upper substantia propria; it was also noted in Patients and methods King Saud University the cytoplasm of fibroblasts, in the walls of Study subjects and specimen collection Riyadh Saudi Arabia blood vessels in the substantia propria, and in School children in a village in the Eastern K. Geboes the walls of accessory lacrimal glands. province of Saudi Arabia were examined using Laboratory of Conclusions Our data indicate new type V a binocular magnifying loupe (X4 Histochemistry and collagen formation, and increased types I, III magnification) and a portable light source. The Cytochemistry and IV collagen content, in the conjunctiva diagnosis of active trachoma and the grading University Hospital from patients with active trachoma. St Rafael system of the intensity of the disease were Leuven performed using the recommended World Belgium Key words Chlamydia, Collagen, Conjunctiva, 1 Health Organization criteria. A total of nine Trachoma L. Missotten children with active trachoma between 7 and 16 Department of years of age were included in the study (mean Ophthalmology Trachoma, a chronic follicular conjunctivitis, age 9.4 years). All patients were asymptomatic University Hospital St Rafael remains a major worldwide infectious cause of and had mild to moderate active trachoma. A Leuven mm blindness, and a major public health problem, 2 X 2 upper palpebral conjunctival biopsy Belgium particularly in developing countries. It is specimen was obtained from each patient. None A.M. Abu EI·Asrar � estimated to affect 500 million people, 7 million of the patients was on topical or systemic Department of 1 of whom are blind. It is caused by serovars A, therapy. The study was approved by the Ophthalmology B, Ba and C of Chlamydia trachomatis? The Research Center, College of Medicine, King King Abdulaziz University Hospital blinding complications of trachoma are Saud University and the patients admitted to Airport Road associated with progressive conjunctival and the study gave their informed consent. PO Box 245 subconjunctival fibrosis that may lead to dry In addition, four upper palpebral Riyadh 11411 eye syndrome, entropion, trichiasis and corneal conjunctival biopsy specimens were obtained Saudi Arabia blindness. The possible factors involved in from patients undergOing strabismus surgery Fax: +966-1-477 5741 Eye (1998) 12, 735-739 © 1998 Royal College of Ophthalmologists 735 without obvious inflammation and served as controls. noted between inflammatory cells in the stromal The control patients were in a similar age group. lymphoid follicles, suggesting an increased production of collagen types I and III. Antibody to type IV collagen showed irregularly thickened epithelial basement Immunohistochemical staining membrane. Patchy anti-type V collagen staining was The conjunctival biopsy specimens were immediately observed in the upper part of the substantia propria. snap-frozen in Tissue-Tek optimum cutting temperature Type V collagen staining was also observed in the (OCT) compound (Miles Laboratories, IN) and cytoplasm of fibroblasts, in the walls of blood vessels in maintained at -80°C until use. For the substantia propria, and in the walls of accessory immunohistochemistry, 5 !Lm serially cut cryostat lacrimal glands (Fig. 2). sections were dried overnight at room temperature, fixed in absolute acetone for 10 min and stained with a three­ Discussion step avidin/biotin peroxidase-labelled complex procedure. Rehydrated slides were incubated for 30 min Biochemical alterations in the connective tissue matrix with the monoclonal and polyclonal antibodies listed in are a common feature of many diseases, and account in Table 1, which were diluted to an optimum major part for their functional impairment. Such concentration. The secondary and tertiary antibodies alterations are especially important in acute and chronic consisted of biotin-conjugated rabbit anti-mouse inflammatory diseases, where they may take the form of immunoglobulin and the avidin/biotin peroxidase­ degradation of matrix components or their excessive labelled complex, respectively, which were both accumulation leading to fibrosis. Histochemically, we purchased from Dakopatts (Copenhagen, Denmark). All observed that the conjunctival stroma from patients with incubations were carried out for 30 min at room active trachoma contained type V collagen. On the other temperature, then washed in three changes of phosphate­ hand, type V collagen was not detected in the normal buffered saline at pH 7.2 for 15 min. The reaction product conjunctiva. This is consistent with the observations of was developed by addition of diaminobenzidine and others who reported accumulation of type V collagen in H 0 , resulting in brown immunoreactive sites. The 2 2 conditions associated with tissue remodelling and new slides were faintly counterstained with Harris collagen synthesis. Increased type V collagen content has haematoxylin. Finally, the sections were rinsed with been detected in other diseased tissues such as cardiac distilled water and coverslipped with glycerol. Controls, hypertrophy,8 neoplasia,9,l0 Crohn's disease,u which were invariably negative, consisted of omission of atherosclerotic lesions,12,13 pseudointima of vascular primary or secondary antibody and use of chromogen grafts7 and chronically inflamed gingival tissue,16 alone. suggesting that it may play an important role in the pathology of these diseases. Type V collagen was first identified in human Results placenta18 and skin extracts.19 It has since been identified In normal conjunctival tissue specimens, the substantia in many tissues, including cornea?,20,21 Some studies propria showed diffuse, fine fibrillar staining with showed a preferential pericellular localisation of collagen antibodies to types I and III collagen. Staining was more V?,22 Due to its pericellular localisation type V collagen intense in a band-shaped area located just underneath may interact with cell surfaces and modulate cellular the epithelium and in the perivascular areas. The activities such as adhesion, differentiation, migration or epithelial and capillary endothelial basement membranes synthesis. Several cell lines including fibroblasts were reacted with type IV collagen antibody. No staining was found to adhere and spread on native and denatured observed with antibody to type V collagen (Fig. 1). type V collagen?3 Cell-surface-associated heparan Trachoma specimens showed abnormal deposition of sulphate appears to mediate the attachment of cells to fibrils of types I and III collagen among epithelial cells. type V collagen?4 In addition, type V collagen has been Patchy increase in staining intensity with antibodies to postulated to play a role in regulating the diameter of types I and III collagen was noted in the substantia heterotypic fibrils composed of type V collagen propria just underneath the epithelium. Thicker and molecules co-assembled along those of type I collagen. irregularly arranged collagen type I and III fibrils were Increased expression of type V relative to type I collagen Table 1. Monoclonal and polyclonal antibodies used in this study Antibody Specificity Working dilution Sourcea Anti-type I collagen (pc) Type I collagen 1:10 Campro Anti-type III collagen (pc) Type III collagen 1:10 Chemicon Anti-type