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Episodic Conjunctival Inflammation after Stevens-Johnson Syndrome
Episodic Conjunctival Inflammation after StevensJohnson Syndrome C. STEPHEN FOSTER, MD,·,2,3 LYE P. FONG, MD/ DIMITRI AZAR, MD,3,4 KENNETH R. KENYON, MD 2,3,4 Abstract: The authors studied the histopathologic, ultrastructural, and immunopathologic characteristics of conjunctiva from patients with Stevens-Johnson syndrome (SJS). A small subset of SJS patients with recurrent conjunctival inflammation unassociated with external factors such as lid margin keratinization , sicca syndrome, trichiasis, or entropion was identified. The ultrastructural and immunopathologic characteristics of the conjunctiva from these patients were distinctly different from those of the conjunctiva from SJS patients without recurrent conjunctivitis, and suggested an active, immunologically mediated inflammation . Vasculitis or perivasculitis, immunoreactant deposition in vessel walls, vascular basement membrane disruption, thickening, and reduplication, and a preponderance of helper T-Iymphocytes, macrophages, and Langerhans' cells were the notable distinguishing features in those patients with recurrent conjunctival inflammation. This rare clinical syndrome may represent the ocular counterpart to recurrent dermal or oral mucosal erythema multiforme. [Key words : conjunctivitis, herpes simplex virus, immune complex, StevensJohnson syndrome, vasculitis.] Ophthalmology 95:453-462, 1988
Erythema multiforme is a complex immunologic syndrome that is characterized, in its mildest form, by typical "target" -type skin lesions. In its most severe form, known as Stevens-Johnson syndrome (SJS), it is a life-threatening disorder that affects mucous membranes including the conjunctiva. The immune complex vasculitis and/or the lymphocyte-dependent effector pathways responsible for the lesions may be precipitated by microbial, neoplastic, or pharmacologic agents. The syndrome is usually an acute, self-limited disorder but a less well-recognized recurrent form of the disease also exists. Fitzpatrick et all described a case of recurrent Originally received: October 15, 1987. Revision accepted: December 16, 1987. Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, Boston. 2 Department of Ophthalmology, Harvard Medical School, Boston. 3 Cornea Service, Massachusetts Eye and Ear Infirmary, Boston. 4 Eye Research Institute of Retina Foundation, Boston. 1
Supported in part by NIH grants EY 06008 (Dr. Foster) and EY 03967 (Dr. Kenyon) . Reprint requests to C. Stephen Foster, MD, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114.
erythema multi forme affecting the skin, and speculated that ultraviolet irradiation periodically induced inflammatory mediator release, thereby stimulating the recurrent skin lesions. Lozada and Silverman 2 reported, in 1978, the clinical characteristics and natural history of recurrent oral erythema multiforme in 50 patients; and Bean and Quezada 3 reported on 11 additional patients with recurrent oral erythema multiforme. These latter authors, in addition to confirming the clinical characteristics described previously by Lozada and Silverman 2 also described factors which seemed to precipitate the recurrent episodes. The primary purpose of this report is to describe the clinical characteristics and the histopathologic and immunopathologic correlates in the conjunctiva of nine patients with a well-documented history of SJS, with subsequently developing recurrent episodes of conjunctival inflammation unassociated with external aggravating factors, and unassociated with skin or nonocular mucosal recurrent manifestations. Histopathologic and immunopathologic studies of conjunctival biopsies from these patients disclosed an immune complex vasculitis. We believe these patients represent a small subset of individuals with a history of SJS who have episodic, 453
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immunologically mediated inflammatory disease of the conjunctiva. The ocular clinical, immunopathologic, and ultrastructural characteristics of these subjects are presented.
MATERIALS AND METHODS Eighteen patients with a history of SJS seen over a I O-year period, from 1977 to 1986, form the basis of this report. The acute, original exanthum must have been characterized by classic target-type erythema multiforme skin lesions in association with acute conjunctivitis. The study was conducted as a prospective one during this 10-year period. The initial study parameters were the clinical features and the histopathologic and immunopathologic correlates. Additional observations were made as the study evolved. Thus, immunoperoxidase, electron microscopy, and circulating immune complex data were not collected on every patient. Nine of the patients experienced recurrent episodic conjunctival inflammation. A cohort population of nine additional individuals with SJS without recurrent episodes of conjunctival inflammation also was studied for comparison. External aggravants included uncorrected sicca syndrome, trichiasis, dystichiasis, entropion, untreated blepharitis/meibomianitis, and keratinization of palpebral conjunctiva adjacent to the eyelid margin. Such aggravating factors were aggressively treated in all patients: trichitic and dystichitic lashes were destroyed by cryoablation; entropion was corrected surgically; keratitis sicca was treated with preservative-free ocular lubricants and/or punctal occlusion. Ocular irritation from tarsal conjunctival keratinization was treated with topical retinoids and with continuous-wear, ultra-thin bandage soft contact lenses. Conjunctival biopsies were obtained, after obtaining informed consent, from all patients 3 months after elimination of the aforementioned confounding variables. Biopsies from the patients with recurrent conjunctivitis were taken during episodes of inflammation and were obtained from bulbar conjunctiva, adjacent to the limbus, typically at the 3-, 9-, or 12-0'clock position, in an area of active inflammation. Normal conjunctival control specimens were obtained from volunteers at the time of cataract surgery. The 4-mm square piece of perilimbal conjunctiva was divided into three pieces for light microscopy, immunofluorescence and immunoperoxidase studies, and transmission electron microscopy. Specimens for light microscopy were fixed overnight in Karnovsky's fixative (I % paraformaldehyde, and 1.25% glutaraldehyde, in 0.2 moljL sodium cacodylate buffer) and transferred to 0.2 moljL sodium cacodylate buffer, which was changed three times at 20-minute intervals. Specimens were then dehydrated, infiltrated with glycol methacrylate solution, and embedded in Historesin. Two micron-thick sections were stained with alkaline Giemsa, periodic acid-Schiff, and hematoxylin-eosin. . Tissue specimens for transmission electron microscopy were immediately fixed in a modified Karnovsky's 454
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fixative, postfixed in osmium tetroxide, dehydrated, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate and were examined with a Phillips 400 transmission electron microscope. Specimens for immunofluorescence were snap frozen, embedded in cryostat embedding compound (OCT compound, Tissue-Tek, Miles Laboratories, Naperville, IL), and stored at -70°C. Four micron-thick sections were air-dried, washed in phosphate-buffered saline, and incubated with fluorescein and rhodamine conjugated rabbit antisera directed against the human immunoglobulins, complement components C3 and C4, fibrinogen, and (as a control for specificity of staining) albumin (Cappel Scientific, Malvern, PA). The slides were washed three times in phosphate-buffered solution (10 minutes each) and then coverslipped with 0.1 % p-phenylenedimine in glycerol and phosphate-bicarbonate buffer (Zeiss). Fluorescence microscopy was performed on a Zeiss Photomic III fluorescence microscope with a vertical illuminator III RS for epifluorescence, a band pass excitation filter (450-490 nm) and barrier filter (520-560 nm) for fluorescein conjugates, and a 515 to 560-nm band pass excitation filter and a long wave pass barrier filter blocking all wavelengths below 590 nm for rhodamine conjugates. Photographic documentation of results was achieved through an on-plane spot metering automatic exposure device. Immunoperoxidase studies were performed using the avidin-biotin complex (ABC) technique employing primary mouse anti-human antibodies for T - and B-cells, peripheral blood monocytes and HLA-Dr (Becton and Dickinson, Mountain View, CA), and Vectastain rabbit anti-mouse kit (Vector Labs, Inc, Burlingame, CA) for secondary and tertiary antibody staining. Antibody Mac-I, a rat antibody that stains tissue macrophages (Hybritech Inc, San Diego, CA), was labeled with a biotinylated rat antibody and standard Vectastain kit. For herpes simplex, a polyclonal rabbit anti-herpes simplex I and II antibody (Dako Corp, Santa Barbara, CA) was used and labeled with a rabbit Vectastain kit. Circulating serum immune complexes were assayed by Raji cell and by CIQ binding assay by acommercial laboratory (Bioscience Labs, Waltham, MA) for the most recent three patients in this study, before and during cytotoxic chemotherapy.
RESULTS Three of the nine patients with recurrent inflammation were male and six were female. The average age at the initial, acute development of SJS was 25 years (range, 7-42 years) and the mean age at onset of episodic inflammation was 22112 years. The average duration of recurrent episodes of conjunctival inflammation was 7 years (range, 3 months to 29 years). A precipitating agent (drug ingestion) for the initial SJS episode was identifiable in only three of the patients. The demographic characteristics of the nonrecurrent SJS patients did not differ significantly from those of the recurrent
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patients. None of the recurrent SJS patients had recurrent skin lesions typical of erythema multiforme; one patient had recurrent oral mucosal ulcers compatible with recurrent erythema multi forme of oral mucosa, but not concurrent with the development of the episodes of conjunctivitis. The frequency of recurrent episodes of conjunctival inflammation, once external ocular confounding variables were eliminated, was every 3 to 12 weeks. The episodes lasted 8 days to 5 weeks. Three patients related the onset of the episodes to emotional stress, two to the onset of menstruation, and two to herpes labialis.
CASE REPORTS Case 1. An idiopathic SJS developed in a 32-year-old woman in May 1982. Her resultant conjunctival cicatrization and sicca syndrome were controlled with ocular lubricants, but her course was punctuated with intense episodic ocular inflammation, controllable to variable degrees with high-dose oral prednisone. When first seen in the Immunology Service at the Massachusetts Eye and Ear Infirmary in January 1983, visual acuity was counting fingers at 2 feet in the right eye, and 20/300 in the left. Intense bulbar and palpebral conjunctival injection was present bilaterally. Conjunctival keratinization, fornix foreshortening, and symblepharon formation were obvious. Extensive corneal neovascularization and scarring were present in the right eye and corneal scarring and neovascularization were present in the inferotemporal quadrant, extending nearly to the visual axis in the left eye. A paracentral epithelial defect and stromal infiltrate were present in the left eye. Results of bulbar conjunctival biopsy of the right eye showed absence of goblet cells, subepithelial lymphocyte and plasma cell infiltrate, and an aggregation of macrophages and lymphocytes around vessels. Immunofluorescence studies showed an intense deposition of IgA on the conjunctival epithelium, in the conjunctival stroma, and in the walls of conjunctival vessels. The fourth component of complement also was deposited on the conjunctival epithelium. CIQ binding and Raji cells assays showed the presence of circulating immune complexes in the patient's serum; the antibodies in these complexes were IgG, IgA, and IgM class. Topical I % prednisolone acetate 16 times daily failed to control the recurrent episodes of conjunctival inflammation. Case 2. An idiopathic SJS developed in a 7-year-old boy in September 1977. Acute treatment was with high-dose oral prednisone and topical Decadron (dexamethasone) ointment to both eyes for 2 weeks. Trichiasis, conjunctival cicatrization, cicatricial entropion, and sicca syndrome developed. Cryoablation of trichitic lashes, ocular lubrication, and bandage soft lens therapy controlled the consequences of scarring from the acute episode. Extensive corneal scarring and neovascularization were present in the left eye because of persistent epithelial defects and microbial keratitis. Paracentral corneal scarring and neovascularization were present in the right eye. The visual acuity in January 1978 was 20/70 in the right eye and 20/200 in the left. Recurrent trichiasis and dystichiasis were managed with cryoablation of lashes. Despite control of the sicca component and the trichiasis, recurrent episodes of intense conjunctival inflammation developed. The degree of stromal keratitis and neovascularization in the right eye increased with each of these episodes. In June 1982, during a recurrent episode of inflammation, conjunctival biopsy of the
left eye was performed. Conjunctival epithelial keratinization, subepithelial lymphocyte and plasma cell infiltration, and perivascular aggregation of lymphocytes and macrophages were noted. Immunofluorescence studies showed IgA and IgD in the vascular walls. A large number of mast cells in varying degrees of degranulation was noted in the substantia propria. Circulating immune complexes were found in the serum by CIQ binding and by Raji cell assay. Recurrent oral mucosal lesions occurred, not in association with recurrent conjunctival inflammation. Case 3. An idiopathic SJS developed in a 20-year-old woman in August 1981. Despite control of sicca syndrome and in the absence of exposure to trichiasis, she was plagued by recurrent episodes of violent bulbar conjunctival inflammation. When seen on January 6, 1983, the visual acuity was 20/100 in the right eye and 20/400 in the left. Bilateral central epithelial defects were present, and peripheral corneal neovascularization was extensive in the left eye. The punctae were closed, and Schirmer values were 5 mm in both eyes (5 minutes after topical anesthetic). No trichiasis or dystichiasis was observed. Results of conjunctival biopsy of the left eye showed keratinization of the conjunctival epithelium, plasma cell and lymphocyte infiltration of the substantia propria, perivascular cuffing with macrophages and lymphocytes, and immunofluorescence disclosed IgG, IgA, IgD, and IgM, the third and fourth components of complement deposited in the vessel walls. Topical 1% prednisolone acetate and retinoic acid 0.01 % failed to control the recurrent episodes of conjunctival inflammation.
LIGHT MICROSCOPIC FINDINGS The light microscopic characteristics of conjunctival specimens from patients with the chronic consequences of a typical single (nonrecurrent) episode of SJS included features of epithelial keratinization, diminished goblet cell population, and a diffuse, mild, chronic mononuclear cell infiltrate in the subepithelial stroma. In no instance was a vasculitis or perivasculitis noted, nor was there any evidence of prominent mast cell participation and degranulation in the substantia propria. In contrast, all specimens from patients with recurrent conjunctival inflammatory episodes showed vascular involvement (Table 1). Specimens from four patients showed classic vasculitis, with inflammatory cell infiltration into the vessel wall, and fibrinoid necrosis of the vascular wall (Fig 1). Specimens from the remaining four patients showed prominent perivasculitis with extreme degrees of inflammatory cell aggregation around vessels; the predominant cell populations included lymphocytes, macrophages, and plasma cells. Squamous metaplasia, with surface keratinization and diminished goblet cell populations also were noted, as were substantial numbers of degranulating mast cells in most specimens.
IMMUNOFLUORESCENT FINDINGS Specimens from eight patients with recurrent conjunctivitis exhibited immunoreactant deposition in
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Table 1. Conjunctival Vascular Immunoreactant Deposition Case No.
IgG
1
2 3
4
5 6 7 8 9
Total positive
IgA
IgD
+ + +
+
+ + + + +
+ + +
5
6
IgM
+ + + +
5
conjunctival vessels (Fig 2; Table 1). Each patient had I or more immunoreactants in the vessel wall and these were in varying combinations in the individual patient. Only one specimen showed albumin; in five specimens, fibrinogen deposition was found in the vessels (data not
IgE
+
C3
+ + + +
0
C4
+
+
+ + + +
5
5
Histologic Findings Vasculitis Perivasculitis Vasculitis Vasculitis Perivasculitis Perivasculitis Vasculitis Perivasculitis Perivasculitis
shown). Vascular IgG deposition can be difficult to discriminate from the normal background IgG staining in normal human conjunctiva; we read as positive for IgG only vessels that were strikingly more fluorescent than the substantia propria and we may, thus, have underes-
Fig 1. Case 2. Top left, section of conjunctival biopsy. Notice attenuated epithelium and the intense mononuclear cell perivasculitis (alkaline Giemsa; original magnification, XI60). Case I. Bottom left, section of conjunctival biopsy. Notice vessel wall invasion with necrosis (hematoxylin-eosin, original magnification, X250). Fig 2. Case I. Top righI, immunofluorescence photomicrograph of conjunctival biopsy after incubation with fluoresceinated anti-lgA antibodies. Notice fluorescence of conjunctival vessel walls, demonstrating 19A deposition in the vessels (original magnification, XSO). Case S. Bottom right, immunofluorescence photomicrograph of conjunctival biopsy after incubation with rhodamine-conjugated anti-C3 antibodies. Notice fluorescence of vessel wall, demonstrating C3 deposition in the vessel (original magnification, X250).
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timated the number of patients with true IgG vascular deposition. It is to be noted that the epithelial basement membrane zone was unequivocally negative for immunoreactant deposition in all instances. Specimens from patients with the typical chronic consequences of SJS (no recurrent conjunctivitis) lacked any evidence of immunoreactant deposition in vessel walls.
Table 2. Mononuclear Cell Subsets in Stevens-Johnson Syndrome Conjunctiva Cell Count* In Normal Controlst Antibody Cells Stained
IMMUNOPEROXIDASE STUDIES Immunoperoxidase studies were performed on conjunctival specimens from the two most recent patients (cases 8 and 9) and from ten normal donors (cataract surgery patients); the results are shown in Table 2. The predominant T -cells in the conjunctiva from SJS patients were T-helpers; suppressor T-cells were conspicuously rare. This is in sharp contrast to normal human conjunctiva, which contains more resident suppressor T-lymphocytes than helper T-cells. There were numerous, prominent HLA-DR-positive staining cells in the substantia propria, vessel walls, and epithelium of the conjunctiva from the SJS patients. Aproportion of these HLA-DR-positive cells in the epithelium were Langerhans' cells, by virtue of their morphology; the others were macrophages (Fig 3) and, to a smaller degree, activated T -cells. Additionally, the immunoperoxidase technique was used to search for the presence of herpes antigen in conjunctival epithelium, conjunctival vessel walls, and cell surface membranes. In case 8, herpes simplex antigen was detected in all three sites (Fig 4). Herpes simplex virus antigen also could be demonstrated in cells located in the substantia propria of another patient's conjunctiva which lacked epithelium (case 7). Neither of these patients exhibited clinically obvious herpes simplex virus ocular disease, and these were not the patients who related recurrent ocular inflammatory episodes to recurrent herpes labialis.
TRANSMISSION ELECfRON MICROSCOPY FINDINGS The ultrastructural characteristics of squamous metaplasia were noted in all specimens. Specifically, increased epithelial tonofilaments and desmosomes, and decreased goblet cells were noted in both the recurrent and nonrecurrent inflammation specimens (Fig 5). Distinctive differences between the two groups, however, were noted. The epithelial basement membrane zone in conjunctival specimens from patients with recurrent conjunctival inflammation associated with SJS was thickened, sometimes fragmented, and frequently duplicated. These findings were not seen in conjunctival specimens from patients with chronic consequences ofSJS.
Leu-1 T-cells Leu-3a&b T-helpers Leu-2a T-suppressors Leu-14 8-cells Mac-1 Macrophages HLA-OR Langerhans ', macrophages, B-cells, and activated T-cells and monocytes
In Patients
Mean ± SO
8.0
9.0
17.3 5.0 14.7 2.7
o o o o
12.7 10.3 60.0 60.0
5.4 ± 2.1 1.2 ± 0.4 4.2 ± 1.1 2.4 ± 0.7 1.3 ± 0.4 2.6 ± 0.9
SO = standard deviation. * Average number of positively staining cells over three high-power fields. t Mean and standard deviation calculated from ten control specimens.
Vascular endothelial cells in conjunctival specimens from patients with recurrent SJS appeared metabolically active, and an electron-dense material was noted in the widened extravascular zones (Fig 6). In addition, marked endothelial swelling was observed in one patient (Fig 7). Vascular basement membrane zone disruption, reduplication, and thickening were notable features in these specimens, and these findings were in sharp contrast to the intact, normal-appearing vascular basement membrane zone in specimens from patients with nonrecurrent SJS (Table 3). Specimens from two patients who were successfully treated for their recurrent attacks of conjunctival inflammation by immunosuppression showed normal vessel ultrastructural characteristics.
DISCUSSION Stevens-Johnson syndrome4 is generally an acute and self-limited disorder, which can produce devastating long-term ocular consequences because of conjunctival scarring and damage to adnexal structures which result in tear insufficiency, conjunctival epithelial squamous metaplasia, trichiasis, dystichiasis, lagophthalmos, epithelial defects, corneal ulceration, and corneal neovascularization. The data presented from this study suggest that recurrent episodes of conjunctival inflammation develop in a small subset of SJS patients despite adequate control of the aforementioned external problems. The immunologic nature of such recurrent lesions in the skin and in the oral mucosa has been described pre457
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Fig 3. Case 8. Top left. conjunctival biopsy. Monoclonal antibodies directed against human HLA class II (HLA-DR) antige'ns used. Notice the large number ofHLA-DR-positive cells in the specimen (immunoperoxidase avidin-biotin; original magnification, X 160). Center left. section prepared from same specimen as in top left. The section has been processed identically with the exception that, as a control for the specificity of the staining detected in the above figure, phosphate-buffered saline has replaced the primary (anti-HLA-DR) antibody. Notice the lack of nonspecific staining (immunoperoxidase avidin-biotin; original magnification, XI60). Case 9. Bottom left. conjunctival biopsy. Monoclonal antibody is directed against HLA-DR antigens. Notice in addition to the vascular positivity, the cluster of HLA-DRpositive cells adjacent to the vessel (immunoperoxidase avidin-biotin; original magnification, XI60). Fig 4. Case 8. Top right. conjunctival biopsy. Anti-herpes antibody was used. Notice the diffuse staining of the epithelium, suggesting the presence of herpes antigen in the epithelium (immunoperoxidase avidin-biotin; original magnification, X250). Bottom right. same specimen as in top right. adjacent section, same field. This section has been processed identically to the section in top right. except that phosphate-buffered saline has been substituted for the primary (anti-herpes) antibody. Notice the lack of epithelial staining (immunoperoxidase avidin-biotin; original magnification, x250).
viously. To the best of our knowledge, this is the first description of this phenomenon in the conjunctiva. Our histopathologic and immunopathologic results suggest (as do the previously published reports relating to recurrent skin and oral mucosal lesions), that episodic showers of circulating immune complexes may result in immune complex deposition in previously damaged "predisposed" vessels, with resultant immune complex 458
vasculitis. The cellular and molecular mechanistic details are undoubtedly more complex than this, and it is likely that the immunopathogenesis of this syndrome derives from a combination of contributions from multiple components of the immunologic repertoire. Systemic immunosuppression appears to eliminate the immune complex vasculitis in these patients (unpublished data).
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Fig 5. Increased epithelial desmosomes and tonofilaments in a patient with recurrent Stevens-Johnson syndrome (transmission electron microscopy [TEM]; original magnification, X3000). Bottom inset. a higher magnification of the desmosomes and tonofilaments (TEM; original magnification, X 17,400). Top inset. the extensive squamous metaplasia, acanthosis, and keratinization (phase contrast microscopy; original magnification, X260).
The earliest histologic change observed in skin lesions of patients with erythema multiforme is a perivasculitis of the superficial dermal vessels which are surrounded by mononuclear cells. 5 The conjunctival specimens of our patients with recurrent SJS showed either a vasculitis or perivasculitis. We also observed, as did Huff et al 5 and Wuepper et al 6 that there were infrequent neutrophils, eosinophils, or basophils. A possible explanation for the prominent absence of neutrophils in the vasculitic process has been proposed by Wuepper et al,6 who suggested that the immune complex deposits peculiar to SJS may bear C3d receptors and hence attract monocytes and lymphocytes which have C3d receptor sites, in contrast to neutrophils which have receptors for C3b.
When very early skin lesions from patients with SJS were examined by immunofluorescence, IgM and C3 deposits in vessel walls were present in over 75% of patients. 7 Other authors also have noted vascular deposition of immunoreactants. 5,8-11 In our patients with recurrent SJS, the immunoreactants found in vessel walls included IgG, IgA, IgD, C3, and C4. The infrequent finding of IgM in our patients may be explained either by differing components of immune complexes in our patients or by local ultrastructural and transport mechanisms in the conjunctiva which favors deposition of smaller molecular weight complexes. In our study, the vascular immunoreactants were seen in different combinations in individual patients and did not correspond to the immunoreactants seen in nonvascular areas; in ad-
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Fig 6. Endothelial cell (E) basement membrane duplication associated with basal laminar disruption and increased metabolic activity of the endothelial cells. Notice the electron-dense material (arrows) between the reduplicated basal laminae in the extravascular space (TEM; original magnification, X9900).
dition, albumin (molecular weight, 60,000) was observed in the vessel walls of only one patient; these observations would suggest that the vascular immunoreactant deposition was specific. The relative proportions of helper and suppressor Tlymphocytes in the biopsied conjunctiva of the two most recent l'atients in our series showed a complete absence of suppressor T -cells, in notable contrast to the predominance of such cells in the resident lymphocyte population of normal conjunctiva. It is possible that herpes virus contributed to this finding in case 8. It also is possible that unusual local immunoregulatory relation460
ships within the conjunctiva contribute to the episodic conjunctivitis/conjunctival vasculitis in these patients. Similar T-cell subset relative proportions have been noted in the synovium in rheumatoid vasculitis. 12 ,13 Salivary gland biopsies from patients with Sjogren's syndrome also show a lymphocytic infiltrate rich in helper T -cells and nearly devoid of suppressor cells. The HLA-DR antigens, widely expressed on cells in rheumatoid synovium, significantly regressed with penicillamine therapy, as did the number of helper/inducer Tcells, with resultant fall in the ratio of helper/suppressor cells. 14- 16 We have not since done biopsies on our pa-
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Fig 7. Marked vascular endothelial cell (E) swelling and deposition of electron-dense material in the extravascular space are noted in a patient with recurrent Stevens-Johnson syndrome. The basal lamina is obscured (TEM; original magnification, X8800).
tients during or after cytotoxic immunosuppressive therapy, but we have observed in three patients the disappearance of detectable circulating immune complexes with such therapy. Margolis et al,17 in the only report we could find on lymphocyte subsets in the skin of patients with erythema multiforme, found cytotoxic/suppressor cells to predominate in the epidermis. However, in the dermis they found that helper/inducer cells and Langerhans' cells formed the vast majority of cells in the inflammatory infiltrate, just as in our specimens; we found no mononuclear cells in the epithelium. Approximately 15 to 79% of cases of SJS affecting the skin occur 1 to 3 weeks after episodes of recurrent herpes simplex labialis or genitalis. 5,7,9 This clinical observation is supported by laboratory studies. 5,17 Two of our patients recalled preceding episodes of herpes simplex labialis; however, such occurrences may be easily over-
looked, especially if they are subclinical. Herpes simplex antigen was demonstrated by immunoperoxidase methods in the conjunctiva of two additional patients in our study. The epithelial staining was diffuse throughout the thickness of the epithelium, and appeared in some cells to be concentrated around the nurlei. Orton et aI, 18 by direct immunofluorescence labeling of herpes simplex, also demonstrated epidermal cell staining in the skin of SJS patients. One other study5 also recovered virus, by culture and electron microscopy, from skin lesions of recurrent herpes-associated SJS. In one of them, the antigen was observed in the epithelium and in the substantia propria (cell associated) and, more notably, in the vessel walls. This would suggest the possibility of recurrent herpes simplex, with HSV immune complex vasculitis, as a cause for some cases of recurrent SJS affecting the conjunctiva. Herpes may be an underrecognized provocateur in these patients. 461
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Table 3. Conjunctival Ultrastructural Characteristics Frequency of Observation in Specimens from Patients with Recurrent and Nonrecurrent Stevens-Johnson Syndrome Characteristics Keratinization Increased desmosomes Epithelial BMZ Unilamellar Duplicated Multilamellar Goblet cells Absent Decreased Normal stromal cellularity Stromal inflammation Intact vascular endothelium Vascular BMZ Thickening Intact Perivascular infiltrates BMZ
Recurrent (n = 3)
Nonrecurrent (n = 2)
3 3
0 0 2
0 0 3
0 1
1 1 1
2 3
0 1
3
0
0
0
2 0
= basement membrane zone.
REFERENCES 1. Fitzpatrick JE, Thompson PB, Aeling JL, Huff C. Photosensitive recurrent erythema multiforme . JAm Acad Dermatol 1983; 9:419-23. 2. Lozada F, Silverman S Jr. Erythema multiforme: clinical characteristics and natural history in fifty patients. Oral Surg Oral Med Oral Pathol 1978; 46:628-36. 3. Bean SF, Quezada RK. Recurrent oral erythema multiforme: clinical experience with 11 patients. JAMA 1983; 249:2810-12. 4. Stevens AM, Johnson FC. A new eruptive fever associated with stomatitis and ophthalmia: report of two cases in children . Am J Dis Child 1922; 24:526-33. 5. Huff JC, Swinehart JM , Weston WL, et al. Immune complexes involving herpes antigen in erythema multiforme. Clin Res 1979; 27 :242A.
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6. Wuepper KD , Watson PA, Kazmiecrowski JA. Immune complexes in erythema multiforme and the Stevens-Johnson syndrome. J Invest Dermatol 1980; 74:368-71. 7. Kazmierowski JA, Wuepper KD. Erythema multiforme: immune complex vasculitis of the superficial cutaneous microvasculature. JInvest Dermatol1978; 71 :366-9 . 8. Bushkell LL, Mackel SE , Jordon RE. Erythema multiforme: direct immunofluorescence studies and detection of circulating immune complexes. J Invest Dermatol 1980; 74:372-4. 9. Howland WW, Golitz LE, Weston WL , Huff JC. Erythema multiforme: clinical, histopathologic, and immunologic study. J Am Acad Dermatol 1984; 10:438-46. 10. Finan MC, Schroeter AL. Cutaneous immunofluorescence study of erythema multiforme: correlation with light microscopic patterns and etiologic agents . JAm Acad Dermatol1984; 10:497-506. 11 . Imamura S, Yanase K, Taniguchi S, et al. Erythema multiforme: demonstration of immune complexes in the sera and skin lesions. Br J Dermatol1980; 102:161-6. 12. Johnell 0 , Hulth A, Henricson A. T-Iymphocyte subsets and HLADR-expressing cells in the osteoarthritic synovialis . Scand J Rheumato11985; 14:259-64. 13. Walters MT, Smith JL, Moore K, et al. An investigation of the action of disease modifying antirheumatic drugs on the rheumatoid synovial membrane: reduction in T lymphocyte subpopulations and HLA-DR and DQ antigen expression after gold or penicillamine therapy. Ann Rheum Dis 1987; 46:7-16. 14. Lindahl G, Hedfors E, Klareskog L, Forsum U. Epithelial HLA-DR expression and T lymphocyte subsets in salivary glands in SjOgren's syndrome. Clin Exp Immunol1985; 61 :475-82. 15. Takaya M, Ichikawa Y, Shimizu H, et al. T lymphocyte subsets of the infiltrating cells in the salivary gland and kidney of a patient with Sjogren's syndrome associated with interstitial nephritis. Clin Exp Rheumatol1985 ; 3:2 59-63. 16. Fox R, Howell F. Oral problems in patients with Sjogren 's syndrome. Scand J Rheumatol [Suppl] 1986; 61:194-200. 17. Margolis RJ, Tonnesen MG, Harrist TJ, et al. Lymphocyte subsets and Langerhans celiS/indeterminate cells in erythema multiforme. J Invest Dermatol 1983; 81 :403-6. 18. Orton PW, Huff JC, Tonnesen MG , Weston WL. Detection of herpes simplex viral antigens in skin lesions of erythema multiforme. Ann Intem Med 1984; 101 :48-50.
Erythema multiforme is a complex immunologic syndrome that is characterized, in its mildest form, by typical "target" -type skin lesions. In its most severe form, known as Stevens-Johnson syndrome (SJS), it is a life-threatening disorder that affects mucous membranes including the conjunctiva. The immune complex vasculitis and/or the lymphocyte-dependent effector pathways responsible for the lesions may be precipitated by microbial, neoplastic, or pharmacologic agents. The syndrome is usually an acute, self-limited disorder but a less well-recognized recurrent form of the disease also exists. Fitzpatrick et all described a case of recurrent Originally received: October 15, 1987. Revision accepted: December 16, 1987. Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, Boston. 2 Department of Ophthalmology, Harvard Medical School, Boston. 3 Cornea Service, Massachusetts Eye and Ear Infirmary, Boston. 4 Eye Research Institute of Retina Foundation, Boston. 1
Supported in part by NIH grants EY 06008 (Dr. Foster) and EY 03967 (Dr. Kenyon) . Reprint requests to C. Stephen Foster, MD, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114.
erythema multi forme affecting the skin, and speculated that ultraviolet irradiation periodically induced inflammatory mediator release, thereby stimulating the recurrent skin lesions. Lozada and Silverman 2 reported, in 1978, the clinical characteristics and natural history of recurrent oral erythema multiforme in 50 patients; and Bean and Quezada 3 reported on 11 additional patients with recurrent oral erythema multiforme. These latter authors, in addition to confirming the clinical characteristics described previously by Lozada and Silverman 2 also described factors which seemed to precipitate the recurrent episodes. The primary purpose of this report is to describe the clinical characteristics and the histopathologic and immunopathologic correlates in the conjunctiva of nine patients with a well-documented history of SJS, with subsequently developing recurrent episodes of conjunctival inflammation unassociated with external aggravating factors, and unassociated with skin or nonocular mucosal recurrent manifestations. Histopathologic and immunopathologic studies of conjunctival biopsies from these patients disclosed an immune complex vasculitis. We believe these patients represent a small subset of individuals with a history of SJS who have episodic, 453
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immunologically mediated inflammatory disease of the conjunctiva. The ocular clinical, immunopathologic, and ultrastructural characteristics of these subjects are presented.
MATERIALS AND METHODS Eighteen patients with a history of SJS seen over a I O-year period, from 1977 to 1986, form the basis of this report. The acute, original exanthum must have been characterized by classic target-type erythema multiforme skin lesions in association with acute conjunctivitis. The study was conducted as a prospective one during this 10-year period. The initial study parameters were the clinical features and the histopathologic and immunopathologic correlates. Additional observations were made as the study evolved. Thus, immunoperoxidase, electron microscopy, and circulating immune complex data were not collected on every patient. Nine of the patients experienced recurrent episodic conjunctival inflammation. A cohort population of nine additional individuals with SJS without recurrent episodes of conjunctival inflammation also was studied for comparison. External aggravants included uncorrected sicca syndrome, trichiasis, dystichiasis, entropion, untreated blepharitis/meibomianitis, and keratinization of palpebral conjunctiva adjacent to the eyelid margin. Such aggravating factors were aggressively treated in all patients: trichitic and dystichitic lashes were destroyed by cryoablation; entropion was corrected surgically; keratitis sicca was treated with preservative-free ocular lubricants and/or punctal occlusion. Ocular irritation from tarsal conjunctival keratinization was treated with topical retinoids and with continuous-wear, ultra-thin bandage soft contact lenses. Conjunctival biopsies were obtained, after obtaining informed consent, from all patients 3 months after elimination of the aforementioned confounding variables. Biopsies from the patients with recurrent conjunctivitis were taken during episodes of inflammation and were obtained from bulbar conjunctiva, adjacent to the limbus, typically at the 3-, 9-, or 12-0'clock position, in an area of active inflammation. Normal conjunctival control specimens were obtained from volunteers at the time of cataract surgery. The 4-mm square piece of perilimbal conjunctiva was divided into three pieces for light microscopy, immunofluorescence and immunoperoxidase studies, and transmission electron microscopy. Specimens for light microscopy were fixed overnight in Karnovsky's fixative (I % paraformaldehyde, and 1.25% glutaraldehyde, in 0.2 moljL sodium cacodylate buffer) and transferred to 0.2 moljL sodium cacodylate buffer, which was changed three times at 20-minute intervals. Specimens were then dehydrated, infiltrated with glycol methacrylate solution, and embedded in Historesin. Two micron-thick sections were stained with alkaline Giemsa, periodic acid-Schiff, and hematoxylin-eosin. . Tissue specimens for transmission electron microscopy were immediately fixed in a modified Karnovsky's 454
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fixative, postfixed in osmium tetroxide, dehydrated, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate and were examined with a Phillips 400 transmission electron microscope. Specimens for immunofluorescence were snap frozen, embedded in cryostat embedding compound (OCT compound, Tissue-Tek, Miles Laboratories, Naperville, IL), and stored at -70°C. Four micron-thick sections were air-dried, washed in phosphate-buffered saline, and incubated with fluorescein and rhodamine conjugated rabbit antisera directed against the human immunoglobulins, complement components C3 and C4, fibrinogen, and (as a control for specificity of staining) albumin (Cappel Scientific, Malvern, PA). The slides were washed three times in phosphate-buffered solution (10 minutes each) and then coverslipped with 0.1 % p-phenylenedimine in glycerol and phosphate-bicarbonate buffer (Zeiss). Fluorescence microscopy was performed on a Zeiss Photomic III fluorescence microscope with a vertical illuminator III RS for epifluorescence, a band pass excitation filter (450-490 nm) and barrier filter (520-560 nm) for fluorescein conjugates, and a 515 to 560-nm band pass excitation filter and a long wave pass barrier filter blocking all wavelengths below 590 nm for rhodamine conjugates. Photographic documentation of results was achieved through an on-plane spot metering automatic exposure device. Immunoperoxidase studies were performed using the avidin-biotin complex (ABC) technique employing primary mouse anti-human antibodies for T - and B-cells, peripheral blood monocytes and HLA-Dr (Becton and Dickinson, Mountain View, CA), and Vectastain rabbit anti-mouse kit (Vector Labs, Inc, Burlingame, CA) for secondary and tertiary antibody staining. Antibody Mac-I, a rat antibody that stains tissue macrophages (Hybritech Inc, San Diego, CA), was labeled with a biotinylated rat antibody and standard Vectastain kit. For herpes simplex, a polyclonal rabbit anti-herpes simplex I and II antibody (Dako Corp, Santa Barbara, CA) was used and labeled with a rabbit Vectastain kit. Circulating serum immune complexes were assayed by Raji cell and by CIQ binding assay by acommercial laboratory (Bioscience Labs, Waltham, MA) for the most recent three patients in this study, before and during cytotoxic chemotherapy.
RESULTS Three of the nine patients with recurrent inflammation were male and six were female. The average age at the initial, acute development of SJS was 25 years (range, 7-42 years) and the mean age at onset of episodic inflammation was 22112 years. The average duration of recurrent episodes of conjunctival inflammation was 7 years (range, 3 months to 29 years). A precipitating agent (drug ingestion) for the initial SJS episode was identifiable in only three of the patients. The demographic characteristics of the nonrecurrent SJS patients did not differ significantly from those of the recurrent
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patients. None of the recurrent SJS patients had recurrent skin lesions typical of erythema multiforme; one patient had recurrent oral mucosal ulcers compatible with recurrent erythema multi forme of oral mucosa, but not concurrent with the development of the episodes of conjunctivitis. The frequency of recurrent episodes of conjunctival inflammation, once external ocular confounding variables were eliminated, was every 3 to 12 weeks. The episodes lasted 8 days to 5 weeks. Three patients related the onset of the episodes to emotional stress, two to the onset of menstruation, and two to herpes labialis.
CASE REPORTS Case 1. An idiopathic SJS developed in a 32-year-old woman in May 1982. Her resultant conjunctival cicatrization and sicca syndrome were controlled with ocular lubricants, but her course was punctuated with intense episodic ocular inflammation, controllable to variable degrees with high-dose oral prednisone. When first seen in the Immunology Service at the Massachusetts Eye and Ear Infirmary in January 1983, visual acuity was counting fingers at 2 feet in the right eye, and 20/300 in the left. Intense bulbar and palpebral conjunctival injection was present bilaterally. Conjunctival keratinization, fornix foreshortening, and symblepharon formation were obvious. Extensive corneal neovascularization and scarring were present in the right eye and corneal scarring and neovascularization were present in the inferotemporal quadrant, extending nearly to the visual axis in the left eye. A paracentral epithelial defect and stromal infiltrate were present in the left eye. Results of bulbar conjunctival biopsy of the right eye showed absence of goblet cells, subepithelial lymphocyte and plasma cell infiltrate, and an aggregation of macrophages and lymphocytes around vessels. Immunofluorescence studies showed an intense deposition of IgA on the conjunctival epithelium, in the conjunctival stroma, and in the walls of conjunctival vessels. The fourth component of complement also was deposited on the conjunctival epithelium. CIQ binding and Raji cells assays showed the presence of circulating immune complexes in the patient's serum; the antibodies in these complexes were IgG, IgA, and IgM class. Topical I % prednisolone acetate 16 times daily failed to control the recurrent episodes of conjunctival inflammation. Case 2. An idiopathic SJS developed in a 7-year-old boy in September 1977. Acute treatment was with high-dose oral prednisone and topical Decadron (dexamethasone) ointment to both eyes for 2 weeks. Trichiasis, conjunctival cicatrization, cicatricial entropion, and sicca syndrome developed. Cryoablation of trichitic lashes, ocular lubrication, and bandage soft lens therapy controlled the consequences of scarring from the acute episode. Extensive corneal scarring and neovascularization were present in the left eye because of persistent epithelial defects and microbial keratitis. Paracentral corneal scarring and neovascularization were present in the right eye. The visual acuity in January 1978 was 20/70 in the right eye and 20/200 in the left. Recurrent trichiasis and dystichiasis were managed with cryoablation of lashes. Despite control of the sicca component and the trichiasis, recurrent episodes of intense conjunctival inflammation developed. The degree of stromal keratitis and neovascularization in the right eye increased with each of these episodes. In June 1982, during a recurrent episode of inflammation, conjunctival biopsy of the
left eye was performed. Conjunctival epithelial keratinization, subepithelial lymphocyte and plasma cell infiltration, and perivascular aggregation of lymphocytes and macrophages were noted. Immunofluorescence studies showed IgA and IgD in the vascular walls. A large number of mast cells in varying degrees of degranulation was noted in the substantia propria. Circulating immune complexes were found in the serum by CIQ binding and by Raji cell assay. Recurrent oral mucosal lesions occurred, not in association with recurrent conjunctival inflammation. Case 3. An idiopathic SJS developed in a 20-year-old woman in August 1981. Despite control of sicca syndrome and in the absence of exposure to trichiasis, she was plagued by recurrent episodes of violent bulbar conjunctival inflammation. When seen on January 6, 1983, the visual acuity was 20/100 in the right eye and 20/400 in the left. Bilateral central epithelial defects were present, and peripheral corneal neovascularization was extensive in the left eye. The punctae were closed, and Schirmer values were 5 mm in both eyes (5 minutes after topical anesthetic). No trichiasis or dystichiasis was observed. Results of conjunctival biopsy of the left eye showed keratinization of the conjunctival epithelium, plasma cell and lymphocyte infiltration of the substantia propria, perivascular cuffing with macrophages and lymphocytes, and immunofluorescence disclosed IgG, IgA, IgD, and IgM, the third and fourth components of complement deposited in the vessel walls. Topical 1% prednisolone acetate and retinoic acid 0.01 % failed to control the recurrent episodes of conjunctival inflammation.
LIGHT MICROSCOPIC FINDINGS The light microscopic characteristics of conjunctival specimens from patients with the chronic consequences of a typical single (nonrecurrent) episode of SJS included features of epithelial keratinization, diminished goblet cell population, and a diffuse, mild, chronic mononuclear cell infiltrate in the subepithelial stroma. In no instance was a vasculitis or perivasculitis noted, nor was there any evidence of prominent mast cell participation and degranulation in the substantia propria. In contrast, all specimens from patients with recurrent conjunctival inflammatory episodes showed vascular involvement (Table 1). Specimens from four patients showed classic vasculitis, with inflammatory cell infiltration into the vessel wall, and fibrinoid necrosis of the vascular wall (Fig 1). Specimens from the remaining four patients showed prominent perivasculitis with extreme degrees of inflammatory cell aggregation around vessels; the predominant cell populations included lymphocytes, macrophages, and plasma cells. Squamous metaplasia, with surface keratinization and diminished goblet cell populations also were noted, as were substantial numbers of degranulating mast cells in most specimens.
IMMUNOFLUORESCENT FINDINGS Specimens from eight patients with recurrent conjunctivitis exhibited immunoreactant deposition in
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Table 1. Conjunctival Vascular Immunoreactant Deposition Case No.
IgG
1
2 3
4
5 6 7 8 9
Total positive
IgA
IgD
+ + +
+
+ + + + +
+ + +
5
6
IgM
+ + + +
5
conjunctival vessels (Fig 2; Table 1). Each patient had I or more immunoreactants in the vessel wall and these were in varying combinations in the individual patient. Only one specimen showed albumin; in five specimens, fibrinogen deposition was found in the vessels (data not
IgE
+
C3
+ + + +
0
C4
+
+
+ + + +
5
5
Histologic Findings Vasculitis Perivasculitis Vasculitis Vasculitis Perivasculitis Perivasculitis Vasculitis Perivasculitis Perivasculitis
shown). Vascular IgG deposition can be difficult to discriminate from the normal background IgG staining in normal human conjunctiva; we read as positive for IgG only vessels that were strikingly more fluorescent than the substantia propria and we may, thus, have underes-
Fig 1. Case 2. Top left, section of conjunctival biopsy. Notice attenuated epithelium and the intense mononuclear cell perivasculitis (alkaline Giemsa; original magnification, XI60). Case I. Bottom left, section of conjunctival biopsy. Notice vessel wall invasion with necrosis (hematoxylin-eosin, original magnification, X250). Fig 2. Case I. Top righI, immunofluorescence photomicrograph of conjunctival biopsy after incubation with fluoresceinated anti-lgA antibodies. Notice fluorescence of conjunctival vessel walls, demonstrating 19A deposition in the vessels (original magnification, XSO). Case S. Bottom right, immunofluorescence photomicrograph of conjunctival biopsy after incubation with rhodamine-conjugated anti-C3 antibodies. Notice fluorescence of vessel wall, demonstrating C3 deposition in the vessel (original magnification, X250).
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timated the number of patients with true IgG vascular deposition. It is to be noted that the epithelial basement membrane zone was unequivocally negative for immunoreactant deposition in all instances. Specimens from patients with the typical chronic consequences of SJS (no recurrent conjunctivitis) lacked any evidence of immunoreactant deposition in vessel walls.
Table 2. Mononuclear Cell Subsets in Stevens-Johnson Syndrome Conjunctiva Cell Count* In Normal Controlst Antibody Cells Stained
IMMUNOPEROXIDASE STUDIES Immunoperoxidase studies were performed on conjunctival specimens from the two most recent patients (cases 8 and 9) and from ten normal donors (cataract surgery patients); the results are shown in Table 2. The predominant T -cells in the conjunctiva from SJS patients were T-helpers; suppressor T-cells were conspicuously rare. This is in sharp contrast to normal human conjunctiva, which contains more resident suppressor T-lymphocytes than helper T-cells. There were numerous, prominent HLA-DR-positive staining cells in the substantia propria, vessel walls, and epithelium of the conjunctiva from the SJS patients. Aproportion of these HLA-DR-positive cells in the epithelium were Langerhans' cells, by virtue of their morphology; the others were macrophages (Fig 3) and, to a smaller degree, activated T -cells. Additionally, the immunoperoxidase technique was used to search for the presence of herpes antigen in conjunctival epithelium, conjunctival vessel walls, and cell surface membranes. In case 8, herpes simplex antigen was detected in all three sites (Fig 4). Herpes simplex virus antigen also could be demonstrated in cells located in the substantia propria of another patient's conjunctiva which lacked epithelium (case 7). Neither of these patients exhibited clinically obvious herpes simplex virus ocular disease, and these were not the patients who related recurrent ocular inflammatory episodes to recurrent herpes labialis.
TRANSMISSION ELECfRON MICROSCOPY FINDINGS The ultrastructural characteristics of squamous metaplasia were noted in all specimens. Specifically, increased epithelial tonofilaments and desmosomes, and decreased goblet cells were noted in both the recurrent and nonrecurrent inflammation specimens (Fig 5). Distinctive differences between the two groups, however, were noted. The epithelial basement membrane zone in conjunctival specimens from patients with recurrent conjunctival inflammation associated with SJS was thickened, sometimes fragmented, and frequently duplicated. These findings were not seen in conjunctival specimens from patients with chronic consequences ofSJS.
Leu-1 T-cells Leu-3a&b T-helpers Leu-2a T-suppressors Leu-14 8-cells Mac-1 Macrophages HLA-OR Langerhans ', macrophages, B-cells, and activated T-cells and monocytes
In Patients
Mean ± SO
8.0
9.0
17.3 5.0 14.7 2.7
o o o o
12.7 10.3 60.0 60.0
5.4 ± 2.1 1.2 ± 0.4 4.2 ± 1.1 2.4 ± 0.7 1.3 ± 0.4 2.6 ± 0.9
SO = standard deviation. * Average number of positively staining cells over three high-power fields. t Mean and standard deviation calculated from ten control specimens.
Vascular endothelial cells in conjunctival specimens from patients with recurrent SJS appeared metabolically active, and an electron-dense material was noted in the widened extravascular zones (Fig 6). In addition, marked endothelial swelling was observed in one patient (Fig 7). Vascular basement membrane zone disruption, reduplication, and thickening were notable features in these specimens, and these findings were in sharp contrast to the intact, normal-appearing vascular basement membrane zone in specimens from patients with nonrecurrent SJS (Table 3). Specimens from two patients who were successfully treated for their recurrent attacks of conjunctival inflammation by immunosuppression showed normal vessel ultrastructural characteristics.
DISCUSSION Stevens-Johnson syndrome4 is generally an acute and self-limited disorder, which can produce devastating long-term ocular consequences because of conjunctival scarring and damage to adnexal structures which result in tear insufficiency, conjunctival epithelial squamous metaplasia, trichiasis, dystichiasis, lagophthalmos, epithelial defects, corneal ulceration, and corneal neovascularization. The data presented from this study suggest that recurrent episodes of conjunctival inflammation develop in a small subset of SJS patients despite adequate control of the aforementioned external problems. The immunologic nature of such recurrent lesions in the skin and in the oral mucosa has been described pre457
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Fig 3. Case 8. Top left. conjunctival biopsy. Monoclonal antibodies directed against human HLA class II (HLA-DR) antige'ns used. Notice the large number ofHLA-DR-positive cells in the specimen (immunoperoxidase avidin-biotin; original magnification, X 160). Center left. section prepared from same specimen as in top left. The section has been processed identically with the exception that, as a control for the specificity of the staining detected in the above figure, phosphate-buffered saline has replaced the primary (anti-HLA-DR) antibody. Notice the lack of nonspecific staining (immunoperoxidase avidin-biotin; original magnification, XI60). Case 9. Bottom left. conjunctival biopsy. Monoclonal antibody is directed against HLA-DR antigens. Notice in addition to the vascular positivity, the cluster of HLA-DRpositive cells adjacent to the vessel (immunoperoxidase avidin-biotin; original magnification, XI60). Fig 4. Case 8. Top right. conjunctival biopsy. Anti-herpes antibody was used. Notice the diffuse staining of the epithelium, suggesting the presence of herpes antigen in the epithelium (immunoperoxidase avidin-biotin; original magnification, X250). Bottom right. same specimen as in top right. adjacent section, same field. This section has been processed identically to the section in top right. except that phosphate-buffered saline has been substituted for the primary (anti-herpes) antibody. Notice the lack of epithelial staining (immunoperoxidase avidin-biotin; original magnification, x250).
viously. To the best of our knowledge, this is the first description of this phenomenon in the conjunctiva. Our histopathologic and immunopathologic results suggest (as do the previously published reports relating to recurrent skin and oral mucosal lesions), that episodic showers of circulating immune complexes may result in immune complex deposition in previously damaged "predisposed" vessels, with resultant immune complex 458
vasculitis. The cellular and molecular mechanistic details are undoubtedly more complex than this, and it is likely that the immunopathogenesis of this syndrome derives from a combination of contributions from multiple components of the immunologic repertoire. Systemic immunosuppression appears to eliminate the immune complex vasculitis in these patients (unpublished data).
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Fig 5. Increased epithelial desmosomes and tonofilaments in a patient with recurrent Stevens-Johnson syndrome (transmission electron microscopy [TEM]; original magnification, X3000). Bottom inset. a higher magnification of the desmosomes and tonofilaments (TEM; original magnification, X 17,400). Top inset. the extensive squamous metaplasia, acanthosis, and keratinization (phase contrast microscopy; original magnification, X260).
The earliest histologic change observed in skin lesions of patients with erythema multiforme is a perivasculitis of the superficial dermal vessels which are surrounded by mononuclear cells. 5 The conjunctival specimens of our patients with recurrent SJS showed either a vasculitis or perivasculitis. We also observed, as did Huff et al 5 and Wuepper et al 6 that there were infrequent neutrophils, eosinophils, or basophils. A possible explanation for the prominent absence of neutrophils in the vasculitic process has been proposed by Wuepper et al,6 who suggested that the immune complex deposits peculiar to SJS may bear C3d receptors and hence attract monocytes and lymphocytes which have C3d receptor sites, in contrast to neutrophils which have receptors for C3b.
When very early skin lesions from patients with SJS were examined by immunofluorescence, IgM and C3 deposits in vessel walls were present in over 75% of patients. 7 Other authors also have noted vascular deposition of immunoreactants. 5,8-11 In our patients with recurrent SJS, the immunoreactants found in vessel walls included IgG, IgA, IgD, C3, and C4. The infrequent finding of IgM in our patients may be explained either by differing components of immune complexes in our patients or by local ultrastructural and transport mechanisms in the conjunctiva which favors deposition of smaller molecular weight complexes. In our study, the vascular immunoreactants were seen in different combinations in individual patients and did not correspond to the immunoreactants seen in nonvascular areas; in ad-
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Fig 6. Endothelial cell (E) basement membrane duplication associated with basal laminar disruption and increased metabolic activity of the endothelial cells. Notice the electron-dense material (arrows) between the reduplicated basal laminae in the extravascular space (TEM; original magnification, X9900).
dition, albumin (molecular weight, 60,000) was observed in the vessel walls of only one patient; these observations would suggest that the vascular immunoreactant deposition was specific. The relative proportions of helper and suppressor Tlymphocytes in the biopsied conjunctiva of the two most recent l'atients in our series showed a complete absence of suppressor T -cells, in notable contrast to the predominance of such cells in the resident lymphocyte population of normal conjunctiva. It is possible that herpes virus contributed to this finding in case 8. It also is possible that unusual local immunoregulatory relation460
ships within the conjunctiva contribute to the episodic conjunctivitis/conjunctival vasculitis in these patients. Similar T-cell subset relative proportions have been noted in the synovium in rheumatoid vasculitis. 12 ,13 Salivary gland biopsies from patients with Sjogren's syndrome also show a lymphocytic infiltrate rich in helper T -cells and nearly devoid of suppressor cells. The HLA-DR antigens, widely expressed on cells in rheumatoid synovium, significantly regressed with penicillamine therapy, as did the number of helper/inducer Tcells, with resultant fall in the ratio of helper/suppressor cells. 14- 16 We have not since done biopsies on our pa-
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Fig 7. Marked vascular endothelial cell (E) swelling and deposition of electron-dense material in the extravascular space are noted in a patient with recurrent Stevens-Johnson syndrome. The basal lamina is obscured (TEM; original magnification, X8800).
tients during or after cytotoxic immunosuppressive therapy, but we have observed in three patients the disappearance of detectable circulating immune complexes with such therapy. Margolis et al,17 in the only report we could find on lymphocyte subsets in the skin of patients with erythema multiforme, found cytotoxic/suppressor cells to predominate in the epidermis. However, in the dermis they found that helper/inducer cells and Langerhans' cells formed the vast majority of cells in the inflammatory infiltrate, just as in our specimens; we found no mononuclear cells in the epithelium. Approximately 15 to 79% of cases of SJS affecting the skin occur 1 to 3 weeks after episodes of recurrent herpes simplex labialis or genitalis. 5,7,9 This clinical observation is supported by laboratory studies. 5,17 Two of our patients recalled preceding episodes of herpes simplex labialis; however, such occurrences may be easily over-
looked, especially if they are subclinical. Herpes simplex antigen was demonstrated by immunoperoxidase methods in the conjunctiva of two additional patients in our study. The epithelial staining was diffuse throughout the thickness of the epithelium, and appeared in some cells to be concentrated around the nurlei. Orton et aI, 18 by direct immunofluorescence labeling of herpes simplex, also demonstrated epidermal cell staining in the skin of SJS patients. One other study5 also recovered virus, by culture and electron microscopy, from skin lesions of recurrent herpes-associated SJS. In one of them, the antigen was observed in the epithelium and in the substantia propria (cell associated) and, more notably, in the vessel walls. This would suggest the possibility of recurrent herpes simplex, with HSV immune complex vasculitis, as a cause for some cases of recurrent SJS affecting the conjunctiva. Herpes may be an underrecognized provocateur in these patients. 461
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Table 3. Conjunctival Ultrastructural Characteristics Frequency of Observation in Specimens from Patients with Recurrent and Nonrecurrent Stevens-Johnson Syndrome Characteristics Keratinization Increased desmosomes Epithelial BMZ Unilamellar Duplicated Multilamellar Goblet cells Absent Decreased Normal stromal cellularity Stromal inflammation Intact vascular endothelium Vascular BMZ Thickening Intact Perivascular infiltrates BMZ
Recurrent (n = 3)
Nonrecurrent (n = 2)
3 3
0 0 2
0 0 3
0 1
1 1 1
2 3
0 1
3
0
0
0
2 0
= basement membrane zone.
REFERENCES 1. Fitzpatrick JE, Thompson PB, Aeling JL, Huff C. Photosensitive recurrent erythema multiforme . JAm Acad Dermatol 1983; 9:419-23. 2. Lozada F, Silverman S Jr. Erythema multiforme: clinical characteristics and natural history in fifty patients. Oral Surg Oral Med Oral Pathol 1978; 46:628-36. 3. Bean SF, Quezada RK. Recurrent oral erythema multiforme: clinical experience with 11 patients. JAMA 1983; 249:2810-12. 4. Stevens AM, Johnson FC. A new eruptive fever associated with stomatitis and ophthalmia: report of two cases in children . Am J Dis Child 1922; 24:526-33. 5. Huff JC, Swinehart JM , Weston WL, et al. Immune complexes involving herpes antigen in erythema multiforme. Clin Res 1979; 27 :242A.
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6. Wuepper KD , Watson PA, Kazmiecrowski JA. Immune complexes in erythema multiforme and the Stevens-Johnson syndrome. J Invest Dermatol 1980; 74:368-71. 7. Kazmierowski JA, Wuepper KD. Erythema multiforme: immune complex vasculitis of the superficial cutaneous microvasculature. JInvest Dermatol1978; 71 :366-9 . 8. Bushkell LL, Mackel SE , Jordon RE. Erythema multiforme: direct immunofluorescence studies and detection of circulating immune complexes. J Invest Dermatol 1980; 74:372-4. 9. Howland WW, Golitz LE, Weston WL , Huff JC. Erythema multiforme: clinical, histopathologic, and immunologic study. J Am Acad Dermatol 1984; 10:438-46. 10. Finan MC, Schroeter AL. Cutaneous immunofluorescence study of erythema multiforme: correlation with light microscopic patterns and etiologic agents . JAm Acad Dermatol1984; 10:497-506. 11 . Imamura S, Yanase K, Taniguchi S, et al. Erythema multiforme: demonstration of immune complexes in the sera and skin lesions. Br J Dermatol1980; 102:161-6. 12. Johnell 0 , Hulth A, Henricson A. T-Iymphocyte subsets and HLADR-expressing cells in the osteoarthritic synovialis . Scand J Rheumato11985; 14:259-64. 13. Walters MT, Smith JL, Moore K, et al. An investigation of the action of disease modifying antirheumatic drugs on the rheumatoid synovial membrane: reduction in T lymphocyte subpopulations and HLA-DR and DQ antigen expression after gold or penicillamine therapy. Ann Rheum Dis 1987; 46:7-16. 14. Lindahl G, Hedfors E, Klareskog L, Forsum U. Epithelial HLA-DR expression and T lymphocyte subsets in salivary glands in SjOgren's syndrome. Clin Exp Immunol1985; 61 :475-82. 15. Takaya M, Ichikawa Y, Shimizu H, et al. T lymphocyte subsets of the infiltrating cells in the salivary gland and kidney of a patient with Sjogren's syndrome associated with interstitial nephritis. Clin Exp Rheumatol1985 ; 3:2 59-63. 16. Fox R, Howell F. Oral problems in patients with Sjogren 's syndrome. Scand J Rheumatol [Suppl] 1986; 61:194-200. 17. Margolis RJ, Tonnesen MG, Harrist TJ, et al. Lymphocyte subsets and Langerhans celiS/indeterminate cells in erythema multiforme. J Invest Dermatol 1983; 81 :403-6. 18. Orton PW, Huff JC, Tonnesen MG , Weston WL. Detection of herpes simplex viral antigens in skin lesions of erythema multiforme. Ann Intem Med 1984; 101 :48-50.