Br J Ophthalmol: first published as 10.1136/bjo.73.4.276 on 1 April 1989. Downloaded from

British Journal ofOphthalmology, 1989, 73, 276-282

Immunopathology of trachomatous conjunctivitis AHMED M ABU EL-ASRAR', JOOST J VAN DEN OORD,2 KAREL GEBOES,2 LUC MISSOTTEN,3 MOHAMED H EMARAH,' AND VALEER DESMET2 From the 'Department of Ophthalmology, Mansoura University Hospital, Egypt; and the 3Department of Ophthalmology and 2Laboratory ofHistochemistry and Cytochemistry, University Hospital St Rafael, Leuven, Belgium

SUMMARY Upper palpebral conjunctival biopsy specimens obtained from eight patients with active trachoma were examined by routine histological and immunohistochemical methods. The epithelium expressed class I major histocompatibility complex (MHC) products throughout and class II MHC products in the superficial layers. The epithelial inflammatory infiltrate consisted of polymorphonuclear leucocytes, macrophages, T lymphocytes, and dendritic cells. In the underly- ing stroma the inflammatory infiltrate was organised as B lymphoid follicles, and there was also a diffuse infiltrate consisting of plasma cells and scattered B lymphoid cells, dendritic cells, T cells, macrophages, and polymorphonuclear leucocytes. Each type of cell has its special location in the tissue. Plasma cells were located on a subepithelial band and as a dense infiltrate round the acini of accessory lacrimal glands. IgA+ plasma cells outnumbered IgG+ cells, whereas IgM+ and IgE+ cells were presence and cell mediated few. Our data provide good evidence for the of both humoral copyright. immune responses and a possible role for autoimmune mechanisms in the conjunctival tissues of trachoma patients.

Trachoma is a chronic keratoconjunctivitis caused by ally inoculated with C. trachomatis these follicles http://bjo.bmj.com/ Chlamydia trachomatis serotype A, B, or C.' It is one were found to consist of B cells, T cells, and of the leading causes of blindness in underdeveloped macrophages,'3 but no data are available from human countries and affects an estimated 500 million material. Therefore, using the monoclonal antibody people.2 technique for the identification of cells involved in C. trachomatis is essentially a pathogen of the immune response we analysed the in-situ immune mucosal surfaces, infecting and replicating within reaction in trachomatous conjunctivitis in humans in epithelial cells.' Ocular with the agent of order to examine more precisely the of C. trachomatis induces both humoral immunity'7 and this condition. on September 29, 2021 by guest. Protected cell mediated immunity."'0 The immune response seems to confer partial protection against subsequent Subjects and methods infection, yet appears also to be responsible for much of the observed and tissue destruction seen SUBJECTS in trachoma." Eight patients presenting with active trachoma seen Analysis of the components of the local immune in the Ophthalmic Outpatient Clinic of Mansoura response occurring in the conjunctival tissues of University Hospital were included in the study. All patients with active trachoma has so far received were examined by a slit-lamp, and corneal and relatively little attention. The is characterised conjunctival changes were recorded. histologically by the formation of large lymphoid The patients comprised four males and four follicles with prominent follicular centres in the females. Their ages ranged from 3 to 9 years, average stroma. 12 In the conjunctiva of monkeys experiment- 5 years. Four patients were diagnosed clinically as trachoma stage 2a, three as trachoma stage 1, and one as Correspondence to Professor L Missotten, Department of Ophthal- trachoma stage 2b according to MacCallan's mology, University Hospital St Rafael, Kapucijnenvoer 7, B3000 classification.'4 All the patients had upper limbal Leuven, Belgium. active pannus. 276 Br J Ophthalmol: first published as 10.1136/bjo.73.4.276 on 1 April 1989. Downloaded from

Immunopathology oftrachomatous conjunctivitis 277

METHODS procedure' with the following monoclonal anti- Upper palpebral conjunctival biopsy specimens were bodies: MT,, MB2, LN2, TAL-1B5, and 3MA134. obtained from all the patients after informed MTI defines all T lymphocytes'6; MB2 defines all B consent. The specimens were immediately fixed for cells, excluding mature plasma cells'6; LN2 identifies two to three hours in B5 fixative composed of: (A) 90 B cells, macrophages, interdigitating reticulum cells, ml distilled water, 6 g mercuric chloride, 2-074 g and Langerhans cells'7; TAL-1B5 is directed against sodium acetate (CH3COONa.3H20); and (B) 37% major histocompatibility complex (MHC) class II formaldehyde solution, pH 5.7. 9 ml of A and 1 ml of (HLA-DR) a chains'8; and 3MA134 defines mono- B were mixed immediately before use. nuclear phagocytes (Pulford et al., unpublished The specimens were then kept in absolute data). methanol until embedded in paraffin. Semiserial An unlabelled, three-step peroxidase- anti- sections were cut and stained with haematoxylin and peroxidase (PAP) procedure' was applied for eosin for routine histology. Serial 6 gm sections were polyclonal antibodies directed against immuno- stained by a three-step indirect immunoperoxidase globulins A, G, M, and E as well as P2-microglobulin copyright.

jFig. 1 (a) Conjunctiva obtained from a patient with trachoma stage

2a showing secondary lymphoid http://bjo.bmj.com/ V follicle containing a large pale follicular centre (arrows) surrounded by a thin lymphocytic J '~~~-~ mantle (arrowheads). Haematoxylin and eosin, x85. (b) MB2 staining. Note that the lymphoidfollicle and surrounding

mantle are composed mostly of on September 29, 2021 by guest. Protected MB2' B lymphocytes. Three-step indirect immunoperoxidase for MB2, x135. Br J Ophthalmol: first published as 10.1136/bjo.73.4.276 on 1 April 1989. Downloaded from

278 A M A El-Asrar, JJ Van den Oord, K Geboes, L Missotten, M H Emarah, and V Desmet

Fig. 2 TAL-IB5 staining shows membranous HLA-DR expression in the superficial epithelial layer. Three-step z indirect immunoperoxidase for TAL-I B5, x385. Jr 'ry?64,:

(b2-m), which is a non-polymorphic part of the MHC Fig. 3 LN2 staining. Note the presence ofLN2+ dendritic class I. cells in the epithelium, in the underlying stroma, and at the interface between epithelium and stroma (arrow heads). Results Three-step indirect immunoperoxidasefor LN2, x 130.

LIGHT MICROSCOPIC FINDINGS All biopsy specimens showed comparable changes. membranous HLA-DR expression was noted mainly The overlying epithelium showed mild to moderate in the superficial epithelial layers (Fig. 2). The , and was infiltrated by a mixed inflamma- inflammatory infiltrate in the epithelium consisted of copyright. tory infiltrate consisting of mononuclear and large numbers of 3MA134+ macrophages, as well as polymorphonuclear leucocytes. In the superficial variable numbers of MT1+ T lymphocytes and poly- epithelial layers variable numbers of Halberstaedter morphonuclear leucocytes. LN2+ HLA-DR+ Prowazek inclusion bodies were seen. dendritic cells were observed in the deeper epithelial In the underlying stroma, the inflammatory layers as well as in the underlying stroma (Fig. 3), infiltrate was organised as lymphoid follicles and as a where they were admixed with MT1+ T lymphocytes, diffuse infiltrate. Variably sized secondary lymphoid 3MA134+ macrophages, and scattered MB2+ http://bjo.bmj.com/ follicles (Fig. la) containing a large, pale follicular lymphoid cells. Occasional dendritic cells were centre surrounded by a lymphocytic mantle were situated at the interface between epithelium and seen throughout the biopsy specimens. The follicular stroma. centre was composed of small and large activated The lymphoid follicles consisted of MB2+ LN2+ cleaved and non-cleaved lymphoid cells, and con- HLA-DR+ B lymphocytes (Fig. lb). In the follicular tained many stainable body macrophages. The centre large 3MA134+ stainable body macrophages surrounding lymphocytic mantle was thinned, and (Fig. 4a), and some MT1+ T cells were observed (Fig. on September 29, 2021 by guest. Protected contained small darkly stained lymphocytes and 4b). lymphocytes of variable size and shape, indicating a In the subepithelial band of plasma cells (Fig. 5a) progressive transformation. IgA+ cells outnumbered IgG+ cells, whereas IgM+ In the area between the follicles and the and IgE+ plasma cells were rare. A similar Ig epithelium, as well as in areas of diffuse inflamma- distribution was found in the plasma cells populating tion, a mixed population of lymphocytes, poly- the accessory lacrimal glands (Fig. 5b). morphonuclear leucocytes, macrophages, and some mast cells and eosinophils was observed. The blood Discussion vessels in these areas were distended and lined by prominent endothelium. Using in-situ immunohistochemical techniques and a Finally, a band of plasma cells was situated directly panel of monoclonal antibodies we have analysed the underneath the epithelium, and a dense infiltration cell populations involved in the immune response in by plasma cells was observed also around the acini of the conjunctiva of patients presenting with active accessory lacrimal glands. trachoma. Distinct compartments involved in humoral and cell mediated immune responses were IMMUNOHISTOCHEMICAL FINDINGS observed. All epithelial cells expressed ,B2-microglobulin; The humoral immune response was represented by Br J Ophthalmol: first published as 10.1136/bjo.73.4.276 on 1 April 1989. Downloaded from

Immunopathology oftrachomatous conjunctivitis 279

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Fig. 4 (a) 3MA134 staining shows large immunoreactive macrophages in thefollicular centre. Smaller 3MA134* macrophages are present also in the surrounding infiltrate and in the epithelium (arrows). Three-step indirect immunoperoxidasefor 3MA 134, x 135. (b) MTi staining. Few MT,+ T lymphocytes are present in the

follicular centre, whereas many T copyright. lymphocytes are present in the surrounding infiltrate. Three-step indirect immunoperoxidase for MT,, x135. http://bjo.bmj.com/ on September 29, 2021 by guest. Protected

large, active lymphoid follicles formed of B lympho- exposed on the dendritic reticulum cells acting as cytes, scattered transformed B lymphoid cells in the antigen presenting cells in the follicular centre. lymphocytic mantle and area between the follicle and Subsequently the B cells undergo transformation, epithelium, and large numbers of plasma cells below leave the follicular centre, and differentiate into the surface epithelium and around the acini of plasma cells, which migrate through the region accessory lacrimal glands. Previous in-vitro between the lymphoid follicles and the epithelium. studies' N have shown that C. trachomatis stimulates That region was found to consist mainly ofT cells and B cells to proliferate and differentiate into dendritic cells, and it thus appears to represent a immunoglobulin-secreting plasma cells. Differentia- suitable microenvironment for the terminal differen- tion to plasma cells is enhanced by T cells, leading to tiation of B lymphocytes into plasma cells. the secretion of large quantities of polyclonal IgA+ plasma cells outnumbered IgG+ plasma cells, immunoglobulins. Our results indicate that the in- whereas IgM+ and IgE+ plasma cells were con- vivo counterpart of this in-vitro response consists of spicuously rare. These findings are in accordance B cells that are stimulated by chlamydial antigens with the detection of type-specific chlamydial IgA Br J Ophthalmol: first published as 10.1136/bjo.73.4.276 on 1 April 1989. Downloaded from

280 A M A El-Asrar, JJ Van den Oord, K Geboes, L Missotten, M H Emarah, and V Desmet

Fig. 5 (a) IgA staining shows many IgA+ plasma cells in a subepithelial band. PAP method for IgA, x 135. (b) IgA staining. Note the presence ofdense accumulation ofIgA' plasma cells .5 around the acini ofaccessory lacrimal glands. PAP methodfor IgA, x135. copyright. http://bjo.bmj.com/ on September 29, 2021 by guest. Protected

and IgG antibodies in the tears of patients with and at the border of both regions. By analogy with trachoma.' Dense accumulations of plasma cells, the skin23 these dendritic cells might be antigen- showing a similar Ig distribution, were seen around loaded Langerhans cells, previously shown to occur the acini of accessory lacrimal glands, suggesting that in the conjunctival epithelium,24"2 which have these glands constitute part of the conjunctival pro- migrated towards the underlying stroma, where they tective . According to Franklin and present their antigens in an HLA-DR restricted Remus2' these IgA+ plasma cells originate from B manner to T lymphocytes. In addition to helper T cells present in the conjunctiva-associated lymphoid cells involved in B cell differentiation, the antigen- tissue (CALT), and are involved in the formation of specific proliferation of T cells induces the develop- secretary IgA, which has been found to play a part in ment of cytotoxic effector T cells.26 The majority of T the defence against chlamydial infection by prevent- lymphocytes present in the epithelium are likely to ing its adherence to epithelial cells.2" belong to the cytotoxic T cell subset engaged in Scattered LN2+ HLA-DR+ dendritic cells were cytotoxicity of infected cells. For the cytotoxic T cells observed in the epithelium, in the underlying stroma, to mediate cytotoxicity they must recognise class I Br J Ophthalmol: first published as 10.1136/bjo.73.4.276 on 1 April 1989. Downloaded from

Immunopathology oftrachomatous conjunctivitis 281

MHC antigen displayed by diseased cells. Dr W F Bodmer, Imperial Cancer Research Fund, London; 3MA134 was a gift from Dr D Y Mason, Nuffield Department of The epithelial cells not only expressed the class I Pathology, Oxford. All polyclonal antibodies were obtained from MHC associated P2-microglobulin, but were also Dakopatts a/s Denmark. found to express HLA-DR antigens at their surface. HLA-DR antigens are present on cells involved in References immune responses, B activated including cells, T 1 Darougar S. The humoral immune response to chlamydial cells, and antigen-presenting cells. The presence of infection in humans. Rev Infect Dis 1985; 7: 726-30. HLA-DR antigens on these cells is associated with 2 Dawson CR, Jones DR, Tarizzo ML. Guide to trachoma control. antigen recognition and presentation to T cells and Geneva: World Health Organisation, 1981: 7. B and 3 Johnson AP. Pathogenesis and immunology of chlamydial the initiation of specific T cell responses.2728 of the genital tract. Rev Infect Dis 1985; 7: 741-5. We have shown that the normal conjunctival 4 Murray ES, Charbonnet LT, MacDonald AB. Immunity to epithelium does not express HLA-DR antigens." Its chlamydial infections of the eye. I. The role of circulatory and expression on the conjunctival epithelial cells in secretary antibodies in resistance to reinfection with guinea pig is due to the release inclusion conjunctivitis. J Immunol 1973; 110: 1518-25. trachoma patients probably of 5 Treharne JD, Dwyer RStC, Darougar S, Jones BR, Daghfous T. interferon-gamma by activated T cells present in the Antichlamydial antibody in tears and sera, and serotypes of epithelium. Interferon-gamma has been shown to chlamydia trachomatis isolated from school children in Southern induce HLA-DR synthesis and expression by a Tunisia. Br J Ophthalmol 1978; 62: 509-15. variety of epithelial cells in vitro29 and is produced 6 Darougar S, Treharne JD, Minassian D, El-Sheikh H, Dines RJ, Jones BR. Rapid serological test for diagnosis of chlamydial by lymphocytes stimulated with C. trachomatis.3' ocular infections. Br J Ophthalmol 1978; 62: 503-8. HLA-DR expression might allow conjunctival 7 Taylor HR, Johnson SL, Prendergast RA, Schachter J, Dawson epithelial cells to present chlamydial antigens to T CR, Silverstein AM. An animal model of trachoma. 11. The cells and thus to enhance the immune response in importance of repeated reinfection. Invest Ophthalmol Vis Sci 1982; 23: 507-15. trachoma. Moreover, this HLA-DR expression may 8 Watson RR, MacDonald AB, Murray ES, Modabber FZ. result in unopposed T cell proliferation and lead to Immunity to chlamydial infections of the eye. III. Presence and perpetuation of the immune response. In addition, duration of delayed hypersensitivity to guinea pig inclusion copyright. induction of HLA-DR expression on epithelial cells conjunctivitis. J Immunol 1973; 111: 618-23. 9 Sacks DL, Todd WJ, MacDonald AB. Cell-mediated immune has been associated with autoimmune reactions -for responses in owl monkeys (Aotus trivirgatus) with trachoma to example, autoimmune thyroiditis3233 and diabetes soluble antigens of Chlamydia trachomatis. Clin Exp Immunol mellitus.34 The epithelial cells expressing HLA-DR 1978; 33: 57-64. might present autoantigens to T lymphocytes which 10 Young E, Taylor HR. Immune mechanisms in chlamydial eye infection: cellular immune responses in chronic and acute activate effector B and T lymphocytes, leading to disease. J Infect Dis 1984; 150: 745-51. induction of an autoimmune reaction."5 In this con- 11 Taylor HR. Ocular models of chlamydial infection. 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