British Journal of Ophthalmology, 1989, 73, 191-196

Immune cells in a case of postherpetic marginal trophiculcerKAREL GEBOES,' AHMED ABU EL-ASRAR,2 AND LUC MISSOTTEN'

From the 'Department of Ophthalmology and 'Laboratory ofHistochemistry and Cytochemistry, UniversityHospital St Rafael, Leuven, Belgium; and the 2Department of Ophthalmology, Mansoura University Hospital,Egypt

SUMMARY The corneal surface was examined by means of replica histology, and the excised limbicconjunctiva was examined by routine histological and immunohistochemical methods withmonoclonal antibodies directed against major histocompatibility class II antigens, lymphocytesubsets, Langerhans cells (HLA-DR, OKT4-Leu3a, OKT8, BA1, B1, and OKT6) and immuno-globulins A, G, M, and D. The findings were compared with those found in normal conjunctiva. Noinflammatory cells were present in the replica of the corneal surface. An inflammatory infiltratecomposed of B lymphocytes and null cells, in addition to T lymphocytes, Langerhans cells, andpolymorphs, was present in the epithelium as well as in the stroma of the limbic conjunctiva. Thecomposition of the infiltrate points towards the involvement of cell mediated immunity as well ashumoral immunity. No immunoglobulins were bound to the conjunctival epithelium.

After prolonged epithelial geographic ulceration orstromal herpetic involvement the surroundingepithelium may fail to grow over the epithelial defecteven in the absence of active viral or secondarybacterial infection.' This failure produces indolentepithelial ulcers which may run a particularly pro-longed course and do not respond to therapy withtopical antiviral drugs. The ulcers are usually compli-cated by profound corneal anaesthesia, and herpessimplex virus cannot be recovered from the cornea.2The precise pathogenesis of the trophic corneal

ulcer is still not clear. According to Kaufman,' thefailure of regrowth of the corneal epithelium is due tolack of epithelial basement membrane. Dohlman4 onthe other hand believes that the basic problem maybe a mitotic arrest and inhibition of cell sliding at theedge of the epithelial defect. This would be related tothe ratio of cyclic AMP and cyclic GMP in the cells. Ithas been suggested that collagenases and proteasessecreted by the diseased epithelium and stromaperpetuate the corneal damage and that the use ofcollagenase inhibitors may reduce the secondaryinflammation.' Moreover, idoxuridine is directlytoxic to the epithelial cells and may be a contributingCorrespondence to Professor Dr L Missotten, Department ofOphthalmology, University Hospital St Rafael, Kapucijnenvoer 7,B 3000 Leuven, Belgium.

factor in the continued failure of the epithelium tocover the defects.,

Cell-mediated autoimmune phenomena have beenreported in chronic herpetic keratitis with thedemonstration of positive inhibition of leucocytemigration in response to corneal rather than viralantigens.7 On the other hand Mondino et al.8suggested the involvement of humoral autoimmunephenomena in these cases owing to the finding ofimmunoglobulins and complement bound to theconjunctival epithelium adjacent to sites of active orpreviously active herpetic corneal disease. Theydetected circulating antibodies to the conjunctivalepithelium in some cases and concluded that auto-immune phenomena may accompany or aggravatethe extensive tissue destruction found in chronicherpetic keratitis.The aim of this work is to study the cellular

composition and distribution of the inflammatoryinfiltrate in a case of marginal postherpetic trophiculcer in order to detect the role of immune mecha-nisms in the pathogenesis of trophic ulcers.

Subjects and methods

A 70-year-old female patient was followed up in theDepartment of Ophthalmology for a dendritic

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Fig. 1 Trophic ulcer located at the cornea periphery. Theulcer is stained with fluorescein and seen through a cobaltblue filter.

herpetic ulcer which progressed to marginal trophiculcer (Fig. 1). She was treated with bromovinyldeoxyuridine (BVDU) and steroid eye drops inaddition to an eyewash hourly with Ringer's lactatesolution. This treatment remained unsuccessful.A corneal replica was then made as described by

Missotten and Maudgal.9 The technique of making acorneal replica is as follows: (a) instill a local anaes-thetic into the eye, (b) keep the eye open with aspeculum and dry the corneal surface by blowing air,(c) apply a thin layer of collodion amyl acetatesolution, 3%-4% w/v, on the corneal surface, (d) drythe solution by blowing air for about 2 minutes, andpeel off the dried membrane with a fine curvedforceps. This membrane is the replica of the cornealsurface, which can be mounted on glass slides formicroscopic study. The limbic conjunctiva adjacentto the ulcer was excised, and the ulcer area wascleaned under local anaesthesia.The excised limbic conjunctiva was divided into

two parts. One part was fixed in Bouin's solution andembedded in paraffin. Semiserial sections were cutand stained with haematoxylin and eosin for routinehistology. The other part was frozen in liquidnitrogen-cooled isopentane and used for immuno-histochemical study. Serial cryostat sections 5 [tmthick were dried overnight at room temperature andsubsequently fixed in absolute acetone for 10minutes. An indirect immunoperoxidase procedureas described by Mason et al."' was applied for thefollowing monoclonal antibodies: OKT4, Leu 3a,

OKT8, B1, BAI, OKT6, HLA-DR, and monoclonalantibodies directed against immunoglobulins A, G,M, and D.The OK series of monoclonal antibodies were

purchased from Ortho Pharmaceutical Co, Raritan,NJ, USA; Leu 3a, BA, and HLA-DR were obtainedfrom Becton-Dickinson, Sunnyvale, CA, USA; B1was obtained from Coulter Electronic, Hialeah, FL,USA; monoclonal antibodies against immuno-globulins were obtained from Dakopatts a/sDenmark.OKT4 and Leu 3a define the helper/inducer T cell

subset" 12 and were applied simultaneously.' OKTsdefines the suppressor/cytotoxic T-cell subset.' B1reacts with all mature B cells.'5 BA1 defines mature Blymphocytes.' HLA-DR is directed against majorhistocompatibility (MHC) class II antigens.' AndOKT6 defines cortical thymocytes, epidermalLangerhans cells, and interdigitating reticularcells. 18

Following incubation with peroxidase-conjugatedrabbit antimouse Ig (Dakopatts a/s, Copenhagen,Denmark) the reaction product was developed in3-amino-9-ethylcarbazole (Aldrich Co., Beerse,Belgium) and H202 according to Graham et al.2'Sections were briefly counterstained with Mayer'shaemalum and mounted in an aqueous medium.A piece of normal conjunctiva obtained from a

patient subjected to cataract surgery was studied ina similar way in order to correlate the findingsobserved in the case of trophic ulcer with thosepresent in normal conjunctiva.

Results

REPLICA HISTOLOGYThe corneal replica was examined unstained by phasecontrast and oblique illumination microscopy. Thecollodion membrane of the replica was then dissolvedin acetone, and the cells removed with the replicawere stained with Giemsa stain and examined by lightmicroscopy.The ulcer floor in the replica showed few necrosed

cells, which were of irregular shape and arrange-ment. Some of them were fused. The ulcer marginwas raised, forming a ridge sloping towards the floor(Fig. 2). It was formed of collections of degeneratedcells, which were small, elongated, or rounded.These cells were partially fused and darkly stained.They had small, deformed dark nuclei. Many cellsoutside the ulcer area were swollen and some werefused. No inflammatory cells were seen in the surfacelayer.

NORMAL CONJUCTIVAIn the epithelium of the normal conjunctiva

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Immune cells in a case ofpostherpetic marginal trophic ulcer

Fig. 2 Replica showing part ofthe trophic ulcer. Note that the ulcer margin is raised over the surface and is sloping towardsthe floor of the ulcer. Oblique illumination microscopy, x 175.

occasional irregular HLA-DR positive cells werepresent between the epithelial cells. Irregular OKT6+cells were less numerous, and OKT8+ lymphocyteswere rare. The substantia propria contained OKT4-Leu 3a+, OKT8+, BAI+, and B1+ lymphocytes. Inaddition scattered irregular OKT6+ cells were found.The positive cells were present mainly in and aroundthe blood vessels near the epithelium. Most of theround cells as well as the irregular cells were HLA-DR positive. The endothelial cells of the small bloodvessels expressed HLA-DR. No immunoglobulinswere expressed on the epithelial cells, but surfaceimmunoglobulins, namely IgA, IgG, I{gM, and IgD,were present on the surface membranes of someround cells in the stroma.

CONJUNCTIVAL BIOPSY OF THE TROPHIC ULCERRoutine microscopy shows the presence of anincreased inflammatory cell infiltrate in the stroma aswell as in the epithelium. The infiltrate is composedof neutrophilic polymorphs and of mononuclear cells(Fig. 3). In the epithelium the inflammatory cells arefew and scattered. In the stroma the infiltrate is moreintense and more diffuse.

Immunohistochemistry reveals a positive staining

T....... w A* .

A**. A.~~~~'WLM::

Fig. 3 Mononuclear and polymorphonuclear leucocyticcellular infiltrate present in the conjunctiva adjacent to thetrophic ulcer. Hand E, x540.

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Fig. 4 Conjunctival biopsy ofthetrophic ulcer. A: the expression ofMHC class II antigen oninterepithelial cells which are eitherLangerhans cells or lymphocytes isdemonstrated. Immunoperoxidase,x800.

Fig. 4A

for HLA-DR on most of the inflammatory cells (Fig.4A). The infiltrate is composed of OKT6+, OKT4-Leu 3a+, OKT8+, BA1+, and Bj+ cells (Fig. 4B). Theyare present in small numbers within the epithelium

Fig. 4 B:B1~.B lymphocytes inbetween the epithelial cells.Immunoperoxidase, x800.

and in higher numbers in the substantia propria (Fig.5). In addition many mononuclear cells not express-ing any immunoreactivity with the monoclonalantibodies used in this study are observed. The

Fig.4B

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Immune cells in a case ofpostherpetic marginal trophic ulcer

Fig. 5 Bl+, B lymphocytes present in the stroma oftheconjunctiva adjacent to the trophic ulcer.Immunoperoxidase, x800.

conjunctival epithelium is not expressing immuno-globulins. Surface immunoglobulins, namely, IgA,IgG, IgM, and IgD, are found bound to the surfacemembranes of some mononuclear cells in theepithelium as well as in the substantia propria.

Discussion

From the replica histology it appears that there are noinflammatory cells in the surface layer of the trophicherpetic ulcer.

Routine microscopy of the conjunctiva adjacent tothe ulcer shows an increased inflammatory infiltratecomposed of mononuclear cells and polymorpho-nuclear leucocytes. The infiltrate is present in theepithelium and in the underlying stroma. Similarfindings were noted in the conjunctiva adjacent toMooren's ulcer,2 22 and several studies suggestedan autoimmune basis for the pathogenesis of thisulcer.2" 23 24

Immunohistochemistry reveals many inflamma-tory cells expressing the major histocompatibilityclass II antigens. Class II histocompatibility antigensare known to be present on many types of cellsinvolved in immune responses, including B cells,activated T cells, some macrophages, and inter-digitating cells. Their presence on these cells isassociated with antigen recognition and presentationto T cells and the initiation of specific B and T cellresponses.25 2h

In comparison with normal conjunctiva, thenumber of OKT6+, Langerhans cells, and of OKT8+,suppressor/cytotoxic T lymphocytes in the conjunc-tival epithelium near the trophic ulcer is increased. Inaddition OKT4-Leu 3a+, helper/inducer T lympho-cytes and BA1+, B1+, B lymphocytes are present inthe epithelium. In the substantia propria the inflam-matory cell infiltrate is even more intense than in theepithelium. It is composed of a mixture of OKT6+,OKT4-Leu 3a+, OKT8+, B1+, and BAI+ cells. Thesecells are also more numerous than in normalconjunctiva.The most striking observation is the presence of

numerous round cells that are not stained with themonoclonal antibodies used and lack specific identi-fying surface markers for either T or B lymphocytes.Accordingly we suggest that many mononuclear cellspresent in the infiltrate are null cells. Because of theirgranular microscopic aspect some of these null cellsare probably natural killer cells. In man natural killercells have the appearance of large lymphocytes withan idented nucleus and prominent granules.2" It hasbeen shown experimentally that natural killer cellsmay have a role in limiting herpes simplex virusinfections.28No immunoglobulins are expressed on the con-

junctival epithelial cells near the trophic ulcer. Onthe other hand Mondino et al.8 detected immuno-globulins bound to the conjunctival epithelium inseven out of 11 patients with chronic herpetickeratitis.Our findings indicate that an inflammatory

reaction is present in the conjunctiva near the trophiculcer. The inflammatory reaction is predominantly areaction of mononuclear cells. The infiltrate is amixture of accessory cells (Langerhans cells), varioussubtypes of T lymphocytes, B lymphocytes, and nullcells. The presence of both T and B lymphocytessuggests an underlying immunological mechanism.There are at least six antigenic glycoproteins at the

surface of the virion that also appear at the surface ofthe infected cell.' Immune mechanisms can there-fore interact not only with the virus but also with cellsinfected by the virus. This reaction is possible even inthe absence of the replicating virus. We suggest thatHSV-induced antigenic changes of the surface mem-branes of the epithelial cells stimulate immunereactions that lead to the development of a persistentulceration.The presence of Langerhans cells and T lympho-

cytes (helper/inducer and suppressor/cytotoxicsubsets) as well as ofB lymphocytes indicates that theunderlying immune mechanisms are complex andthat both cell mediated immunity and humoralimmunity are involved. The presence of numerousnull cells is more difficult to explain. It might be

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related to local steroid therapy, which is known toinfluence the immune response and the expression ofantigens. But some of the unstained cells are proba-bly natural killer cells and could be directly involvedin the development of the ulcer.

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Accepted for publication 25 March 1988.

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