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Herpes Simplex Stromal and Endothelial Keratitis
Herpes Simplex Stromal and Endothelial I(eratitis Granulomatous Cell Reactions at the Level of Descemet's Membrane, the Stroma, and Bowman's Layer LEONARD M. HOLBACH, MD,t·2 RAMON L. FONT, MD,t GOTTFRIED O. H. NAUMANN, MD2
Abstract: Fifty-three (25%) of 215 keratectomy specimens of patients with herpes simplex stromal keratitis displayed granulomatous reactions at the level of Descemet's membrane (50/53), midstroma (13/53), and Bowman's layer (5/53). Using an immunoperoxidase technique, herpes simplex virus (HSV) antigens were detected in keratocytes, endothelial cells, and foci of epithelioid histiocytes and multinucleated giant cells around Descemet's membrane. Both granulomatous reactions and HSV antigens were identified significantly more often in specimens with ulcerative necrotizing stromal keratitis than in those from patients with stromal scarring or nonulcerative nonnecrotizing keratitis (P < 0.00001 and P < 0.005, respectively). Herpes simplex virus antigens also were present in endothelial cells adjacent to foci of granulomatous reactions around Descemet's membrane in association with disciform stromal scarring. To our knowledge, this is the first demonstration of HSV antigens in human corneal endothelial cells and in the granulomatous reactions at the level of Descemet's membrane. Ophthalmology 1990; 97:722-728
Originally received: November 27, 1989. Revision accepted: February 8, 1990. Cullen Eye Institute, Baylor College of Medicine, Houston. Department of Ophthalmology and Eye Hospital, University Erlangen-Numberg. Dr. Holbach is currently a Fellow of the Deutsche Forschungsgemeinschaft at the Cullen Eye Institute, Baylor College of Medicine, Houston.
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Presented in part at the Association for Research in Vision and Ophthalmology Annual Meeting, Sarasota, 1989, and as a poster at the American Academy of Ophthalmology Annual Meeting, New Orleans, OCl/Nov 1989. Supported by grants from the Deutsche Forschungsgemeinschaft (Ho 1111/1-1, 1·2), West Germany, the Retina Research Foundation, the Lions Eye Bank, Houston, Texas, and Research to Prevent Blindness, Inc, New York, New York. Reprint requests to Ramon L. Font, MD, Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
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Herpes simplex corneal disease is the leading infectious cause of unilateral blindness in the industrialized countries. 1 Its pathogenesis and optimal management remain controversial. Clinical and experimental studies have suggested that herpetic stromal keratitis is probably a result of viral infection and immunologic mechanisms although the exact roles of each have not been completely defined. 2 - 5 Granulomatous cell reactions of the cornea may constitute a common complication in stromal keratitis which may lead to scarring and perforation. If they are centered around Descemet's membrane, they may be recognized clinically.6 It has been suggested that the granulomatous reactions around Descemet's membrane are nonspecific as they have been observed in various conditions. 7,s Although corneal granulomatous cell reactions already have
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been reported in the first half of this century9 and later described in more detail,IO-14 their nature is still controversial. In two previous studies, herpes simplex virus (HSV) antigens were not found in the foci of granulomatous reactions at the level of Descemet's membrane. 15.16 Clinical observations,17,18 experimental studies, 19-22 HSV isolation 23 and its immunofluorescent demonstration in the anterior chamber4.25 suggested a role of the corneal endothelium in the pathogenesis of herpetic stromal keratitis and keratouveitis. To our knowledge, however, the presence of HSV in human corneal endothelium has not yet been documented. This article studies the distribution of granulomatous reactions in herpetic stromal keratitis and determines if herpes simplex antigens occur in human corneal endothelial cells as well as in the epithelioid cells of the granulomatous reactions centered around Descemet's membrane.
MATERIALS AND METHODS Two hundred fifteen corneal buttons were obtained by penetrating (207/215 or 96.3%) or lamellar keratoplasty (8/215 or 3.7%) from patients with a previous clinical diagnosis of herpes simplex stromal keratitis and a definite history of dendritic epithelial or recurrent herpetic stromal disease. Preoperatively, the patients presented with clinically inactive stromal scarring, nonulcerative or ulcerative necrotizing stromal keratitis. Inactive disease for at least 6 months was defined using clinical criteria (e.g., corneal scarring in a white and quiet eye without evidence of epithelial keratitis, recurrent stromal disease, anterior chamber reaction, or mild anterior uveitis). The keratectomy specimens were fixed in 10% buffered formalin. If localized lesions were present, sections were taken through the lesions in order to find areas that were representative of the pathologic changes noted or suspected from the clinical and gross examinations. Multiple sections, cut at 5 JLm from paraffin-embedded tissues, were available from each specimen. Hematoxylin-eosin and periodic acid-Schiff stains were routinely performed. Special stains including Brown and Hopps (modified Gram stain), Grocott's methenamine silver, and Ziehl-Neelsen for acid-fast bacilli were used when indicated. The sections were examined for the presence of granulomatous cell reactions at the level of Bowman's layer, the midstroma or Descemet's membrane. The distribution of the granulomatous reactions was analyzed according to their location centrally, paracentrally, or peripherally. Sections adjacent to those containing granulomatous reactions were deparaffinized by placing the slides in the oven at 51°C for 2 hours followed by immersion in xylene and hydration in decreasing ethanol concentrations. Endogenous peroxidase activity was neutralized using 100% methanol and 0.4% hydrogen peroxide. Nonimmunologic binding of antibodies was blocked by incubation with normal serum (Vectastain avidin-biotin complex [ABC], horse for anti-IgG, and goat for anti-IgM secondary antibodies). Incubation with all primary antibodies was car-
ried out in a moist chamber at 37°C for 60 minutes. The immunohistochemical stainings for HSV-1 antigens, UCHL-1 (mature activated T-cells, subpopulation of resting T-cells within CD4 and CD8 subsets), LN-2 (Blymphocytes), and Leu-7 (natural killer cells) antigens were carried out using commercially available versions of the avidin-biotinylated peroxidase method (Vectastain ABC, for rabbit and mouse IgG as well as mouse IgM [Vector Laboratories, Burlingame, CA]), as previously described. 26 Herpes simplex virus antigens were typed as specific to HSV -1 using monoclonal antibodies directed against epitopes on the ICP 4 and ICP 6 proteins27 of HSV-l (courtesy of Dr. H.B. Qavi, Department of Molecular Virology, Baylor College of Medicine, and Dr. M. Zweig, National Cancer Institute). Specificity and sensitivity of the viral antisera were determined by applying them to human tissues that were culture-proven positive for HSV-l. There was no cross-reactivity with cytomegalovirus antigens. Sections of human thymus and tonsils provided the sensitivity and specificity controls for the demonstration ofUCHL-1, LN-2, and Leu-7 antigens. In control series, the primary antibodies were omitted or substituted by normal rabbit serum or unrelated IgG and IgM mouse monoclonal antibodies. Negative tissue controls included keratectomy specimens with stromal dystrophies from patients without a history of ocular herpes simplex infection. False-positive reactions as seen on edges of the sections were excluded from evaluation. The primary antibodies to HSV -1 (Macintyre strain, lot c 77, 1: 600, ABC) and UCHL-1 (lot 038, 1:20) were from Dako, Santa Barbara, CA. The murine monoclonal anti-antibodies to Leu-7 (lot 0425, prediluted) and LN-2 (lot 8012 B, prediluted) were obtained from Becton Dickinson, Mountain View, CA, and Techniclone International, Santa Ana, CA. Horse anti-mouse IgG (HSV-1, UCHL-1, LN-2; ABC), goat anti-mouse IgM (Leu-7; ABC), and goat anti-rabbit IgG (HSV; ABC) were used as secondary antisera. The sections were incubated with these reagents in a moist chamber at 37°C for 30 minutes. Subsequent application of the avidin-biotinylated peroxidase complex (Vectastain) was carried out under the same conditions for 45 minutes. The sites of peroxidase activity were visualized by incubation offreshly prepared 3,3'-diaminobenzidine tetrahydrochloride (Fluka, Buchs, Switzerland), 5 mg in 10 ml of 0.05 M trishydroxymethylaminomethane buffered saline, pH 7.6, containing 0.0015 % hydrogen peroxide. The slides were counterstained with Gill's hematoxylin, dehydrated, cleared in xylene, and mounted in Histoclad medium (c. Adams, Parsippany, NJ). Statistical analyses for comparing data were performed with the chi-square test.
RESULTS CLINICOPATHOLOGIC CORRELATIONS
Fifty-three (25%) of 215 keratectomy specimens from patients with a clinical diagnosis of herpes simplex stromal
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Fig 1. Top left. central corneal ulcer with descemetocele and impending perforation in a 73-year-old patient who had a history of herpetic stromal keratitis for I y ear. Folds at the level of Descemet's membrane radiate superiorly from the center of the ulcer. Fig 2. Top right. paracentral view of section of the penetrating keratectomy specimen from the patient shown in Figure I. Herpes simplex viral antigens (brown reaction product) are detected in the deeply located stromal keratocytes adjacent to the ulcer with descemetocele (on the right. not shown in this field) (avidin-biotin complex [ABC] method hematoxylin counterstain ; original magnification, X64). Fig 3. Second row left. highpower view of the descemetocele of the same patient shown in Figure I illustrates thinning and splitting of Descemet's membrane with herpes simplex antigens (brown reaction product) on its anterior surface as well as in a small multinucleated giant cell (arrow) clinging to this membrane (ABC method, hematoxylin counterstain; original magnification. X 1(0). Fig 4. Second row right. keratectomy specimen from a 46-year-old man who had recurrent herpetic stromal disease for 15 years followed by a central corneal ulcer. This paracentral view (adjacent to the ulcer) displays deep stromal scarring with scattered neutrophils. Herpes simplex antigens are readily identified in the deep stromal keratocytes. Notice that the endothelial cells do not express detectable viral antigens (ABC method. hematoxylin counterstain ; original magnification , X I00). Fig 5. Third row left. keratectomy specimen from a 2 3-year-old woman who had recurrent herpetic stromal disease for 6 years followed by a central perforated corneal ulcer. Highpower view displays herpes simplex antigens (brown reaction product) in epithelioid
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keratitis and a history of dendritic or recurrent herpetic stromal keratitis displayed granulomatous cell reactions. The 53 patients included 35 males and 18 females (1.9: 1). Their ages at the time of surgery ranged from 10 to 88 years (median, 54 years). Two of 53 patients (3.8%) with granulomatous reactions had bilateral herpetic keratitis. The 53 corneal buttons were obtained exclusively by penetrating keratoplasty. Forty-five (85%) of the 53 patients had undergone a single keratoplasty, four had had two keratoplasties and two had been grafted three times. One patient had had a lamellar keratoplasty followed by a penetrating keratoplasty and two had had only a lamellar keratoplasty. Of the 53 keratectomy specimens, 22 were from patients with ulcerative necrotizing keratitis (Figs 1-6) and 31 from patients with stromal scarring or nonulcerative keratitis (Figs 7-10). Granulomatous reactions were identified significantly more often in specimens from patients with ulcerative necrotizing keratitis (22/38 or 58%; Figs 1-6) than in those with stromal scarring or nonulcerative keratitis (31/177 or 17%; Figs 7-10) (P < 0.00001). All 13 patients with ulcerative necrotizing keratitis who had had corneal perforation showed histopathologically a granulomatous reaction around Descemet's membrane (Fig 5). Corneal granulomatous reactions. Granulomatous reactions were identified histopathologically mostly in the deep stroma and around Descemet's membrane, centrally (50/53 or 94%; Figs 3,5, 10). In 25% (13/53), they were observed in the midstroma and in 9% (5/53) at the level of Bowman's layer. Fifteen keratectomy specimens (28%) displayed granulomatous reactions with simultaneous involvement of Descemet's membrane, the midstroma, and/or Bowman's layer. The granulomatous reactions were located mostly in the central cornea (66% or 35/53; Figs 3, 5, 10), less frequently in the paracentral region (21% or 11/53), or exclusively in the peripheral cornea (13% or 7/53). Lymphocytes that were entrapped in the granulomatous cell foci stained positively for UCHL-1, but negatively for LN-2 and Leu-7 antigens. CLINICO-IMMUNOPATHOLOGIC CORRELAnONS
Immunocytochemical studies of HSV antigens were performed on sections from 58 corneal buttons that had been obtained by penetrating keratoplasty.
Herpes simplex virus antigens were identified significantly more often in keratectomy specimens from patients with ulcerative necrotizing keratitis (91 % or 20/22; Figs 1-6) than in those with non ulcerative nonnecrotizing stromal keratitis and stromal scarring (11 % or 4/36; Figs 7-10) (P < 0.005). Herpes simplex virus antigens were demonstrated more commonly in avascular (92% or 22/24) than in vascularized corneal lesions (8% or 2/24) (P < 0.005). Ulcerative necrotizing stromal keratitis. Twenty (91 %) of 22 keratectomy specimens from patients with ulcerative necrotizing stromal keratitis displayed HSV antigens. All the patients had a deep stromal ulcer at the time of surgery (Fig 1). Thirteen (59%) of the 22 patients presented with corneal perforation. Herpes simplex virus antigens were found mostly in stromal keratocytes and the extracellular stroma (20/20 or 100%; Figs 2, 4), less commonly in endothelial cells (3/20 or 15%; Fig 6), epithelial cells (3/20 or 15%), or epithelioid histiocytes and multinucleated giant cells (3/20 or 15%; Figs 3, 5). Herpes simplex virus antigens were identified mostly within the central portion of the corneal buttons (15/20 or 75%), and less frequently in the peripheral third (3/20 or 15%) or in both central and peripheral portions (2/20 or 10%). The antigens were distributed more often in the deep corneal stroma (14/20; Figs 2, 4)-particularly along Descemet's membrane and the endothelial cells-than in the superficial stroma (2/20) or in both (4/20). Nonulcerative nonnecrotizing stromal keratitis and disciform stromal scarring. Four (11 %) of the 36 keratectomy specimens with non ulcerative nonnecrotizing stromal keratitis or disciform stromal scarring showed HSV antigens. Preoperatively, these four patients had predominantly an avascular scar and thinned stroma centrally (Fig 7). By slit-lamp examination, irregularities at the level of Descemet's membrane (Fig 7) were noted in two patients. The latter were found to correspond histopathologically to granulomatous reactions at the level of Descemet's membrane. Clinically, there was no evidence of epithelial or stromal keratitis or anterior chamber reaction. None of these patients had a recurrence during the last 6 months. Two patients were without any treatment during that time;
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histiocytes as well as small multinucleated giant cells located at the level of the artifactually detached Descemet's membrane. A focal break in Descemet's membrane is readily observed (ABC method, hematoxylin counterstain; original magnification, X 100). Fig 6. Third row right, highpower view of area located more peripherally to that seen in Figure 5. Herpes simplex viral antigens are detected in corneal endothelial cells. The stromal keratocytes do not stain for herpes simplex antigens (ABC method, hematoxylin counterstain; original magnification, X 100). Fig 7. Fourth row left, left cornea of a 10-year-old girl with a central disciform scar with thinning of the stroma. She had a history of herpetic stromal keratitis for 6 years, with no evidence of recurrence during the last 16 months. Notice the opacities at the level of Bowman's layer and the irregularities at the level ofthe endothelium. Visual acuity was 20/100. Fig 8. Fourth row right, paracentral view of section of the corneal button obtained by penetrating keratoplasty from the patient shown in Figure 7. The corneal epithelium is moderately acanthotic. Bowman's layer is focally interrupted. The adjacent superficial stroma shows numerous plump histiocytes containing periodic acid-Schiff (PAS)-positive granules. The stroma centrally is thinned and scarred (on the right) (PAS; original magnification, X40). Fig 9. Bottom left, section of cornea from the same patient shown in Figure 7 displays stromal scarring. Herpes simplex viral antigens are identified exclusively in endothelial cells adjacent to a focus of granulomatous reaction at the level of Descemet's membrane (not seen in this field). Notice that the stromal keratocytes in the overlying scarred stroma do not express detectable viral antigens (ABC method, hematoxylin counterstain; original magnification, X 160). Fig 10. Bottom right, keratectomy specimen from a 29-yearold man with disciform stromal scarring. He had recurrent herpetic stromal keratitis for II years. There was no evidence of recurrence during the last 8 months. There is a florid granulomatous reaction with focal rupture of Descemet's membrane (PAS; original magnification, X 100).
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one of them had topical low-dose steroids (once daily) and acyclovir ointment (once daily), which was discontinued 2 months before surgery, and the fourth patient had topical low-dose steroids (once daily) until 3 months before surgery. Histopathologically, the four HSV-I-positive keratectomy specimens displayed central stromal scarring (Fig 8) with granulomatous foci around Descemet's membrane as well as in the stroma. Herpes simplex virus type 1 antigens were identified in corneal endothelial cells (3/4; Fig 9) and in epithelioid histiocytes and multinucleated giant cells 0/4). The HSVpositive endothelial cells were located adjacent to the foci of granulomatous cell reaction at the level of Descemet's membrane.
DISCUSSION In this clinicopathologic study of 215 keratectomy specimens from patients with herpes simplex keratitis, the presence of HSV antigens was demonstrated in corneal endothelial cells, epithelioid histiocytes and multinucleated giant cells located at the level of Descemet's membrane. To the best of our knowledge, this observation has not been documented previously. Of clinical interest is the expression of HSV antigens in all corneal cell types including endothelial cells. Herpetic epithelial keratitis is known to result from replicating HSV in epithelial cells.28,29 Herpes virus particles and antigens have been identified in stromal keratocytes in both clinically active and inactive disease. 30- 33 The immunocytochemical identification ofHSV antigens in corneal endothelial cells is consistent with experimental evidence showing that HSV replicated in cultured rabbit corneal endothelial cells. 22 Herpes simplex virus antigens also have been demonstrated in rabbit corneal endothelial cells after topical and intracameral application of different strains of HSV. 19,20 In our study, HSV antigens were detected more often in keratocytes than in endothelial cells. These observations parallel the results of experimental studies showing that cultures of rabbit keratocytes in vitro produce higher amounts of infectious virus than those of rabbit endothelial cells. 22 Clinical observations correlating the area of endothelial cell damage to the area of retrocorneal precipitates in herpetic keratouveitits l7 ,18 provided indirect evidence suggesting that involvement of the corneal endothelium may play a significant role in ocular herpetic disease. Herpes simplex virus isolation from the anterior chamber has been documented in patients with anterior uveitis lacking active epithelial or stromal keratitis.23 Our study clearly documents direct involvement of the corneal endothelium in both herpetic disciform stromal scarring and ulcerative necrotizing keratitis. Our results lend support to clinical observations suggesting the promising role of systemic antiviral treatment in deep herpetic keratitis. 34- 36 Since a randomized, controlled, double-masked clinical trial to evaluate the efficacy of oral 726
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acyclovir has not yet been performed, further clinical studies are needed on the judicious administration of topical and systemic antiviral agents in the treatment of herpes simplex stromal and endothelial keratitis. The demonstration of HSV antigens in deep stromal keratocytes and endothelial cells shows clearly that these cells can be targets of the HSV. Furthermore, this provides additional evidence in favor of performing a penetrating corneal graft over a lamellar keratoplasty in the surgical management of herpes simplex corneal disease. Granulomatous reactions of the cornea (Descemet's membrane, stroma and Bowman's layer) were shown to constitute a common tissue response in herpes simplex stromal keratitis, both in the necrotizing and nonnecrotizing types. Their real incidence is probably even higher since we did not routinely perform serial sections from every specimen. The distribution pattern for HSV antigens and the location of the foci of granulomatous reactions was similar in the keratectomy specimens studied. Both occurred throughout all corneal layers involving predominantly the central, deep stroma, adjacent to Descemet's membrane and the neighboring endothelial cells. Their common location in the deep stroma may be related to the stage of the disease at the time of grafting and the previous topical application of antiviral agents and/or steroids with insufficient penetration and concentration of these drugs at the site of activity. All 13 patients in our study who presented with ulcerative necrotizing herpes simplex keratitis and perforation showed histopathologically HSV-associated granulomatous reactions centered around Descemet's membrane. The formation of a granulomatous reaction around Descemet's membrane often precedes corneal perforation in ulcerative necrotizing keratitis. 6 In this situation, its clinical recognition 6,37 may represent an indication for penetrating keratoplasty. The granulomatous host tissue response in herpes simplex keratitis may be regulated by specific nucleotide sequences of HSV 38 or represent a characteristic tissue response regarding the immunologic status of the patient. Possible targets of the granulomatous reactions in herpes simplex corneal disease include the presence of viral antigens as well as cellular and/or basement membrane structures from the host (e.g., Descemet's membrane) which may become antigenically altered by the virus or the disease process, or both. Our findings suggest that a T-cell-mediated immunity plays a major role in the pathogenesis of granulomatous reactions in herpes simplex keratitis. The identification of HSV antigens in the stromal keratocytes and adjacent granulomatous foci of keratectomy specimens from patients who preoperatively had clinically inactive stromal scarring is consistent with results of in vitro cultural isolation of HSV from patients with clinically inactive herpetic stromal scarring. 39-4 I Previous reports42 and our current findings suggest that the clinical impression of "inactive" stromal scarring is not completely reliable, since histopathologic examination ofkeratectomy specimens from these cases demonstrates lymphocytic infiltrates and/or granulomatous foci associated with the presence of HSV antigens. The virus may persist
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within some cells in a slowly replicating form as a lowgrade chronic infection and provoke a mitigated host tissue response. Animal models have provided evidence of a persistent/latent type of infection in peripheral nonneural tissues (skin and cornea).43-46 The presence of HSV antigens and granulomatous reactions was significantly correlated with the necrotizing type of herpetic stromal keratitis. This important clinicopathologic correlation as well as the immunocytochemical identification ofHSV antigens in endothelial cells ofkeratectomy specimens from patients with both ulcerative necrotizing and nonnecrotizing disciform stromal keratitis may provide a further basis for the diagnosis and management of herpes simplex stromal and endothelial disease.
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357-64.
Abstract: Fifty-three (25%) of 215 keratectomy specimens of patients with herpes simplex stromal keratitis displayed granulomatous reactions at the level of Descemet's membrane (50/53), midstroma (13/53), and Bowman's layer (5/53). Using an immunoperoxidase technique, herpes simplex virus (HSV) antigens were detected in keratocytes, endothelial cells, and foci of epithelioid histiocytes and multinucleated giant cells around Descemet's membrane. Both granulomatous reactions and HSV antigens were identified significantly more often in specimens with ulcerative necrotizing stromal keratitis than in those from patients with stromal scarring or nonulcerative nonnecrotizing keratitis (P < 0.00001 and P < 0.005, respectively). Herpes simplex virus antigens also were present in endothelial cells adjacent to foci of granulomatous reactions around Descemet's membrane in association with disciform stromal scarring. To our knowledge, this is the first demonstration of HSV antigens in human corneal endothelial cells and in the granulomatous reactions at the level of Descemet's membrane. Ophthalmology 1990; 97:722-728
Originally received: November 27, 1989. Revision accepted: February 8, 1990. Cullen Eye Institute, Baylor College of Medicine, Houston. Department of Ophthalmology and Eye Hospital, University Erlangen-Numberg. Dr. Holbach is currently a Fellow of the Deutsche Forschungsgemeinschaft at the Cullen Eye Institute, Baylor College of Medicine, Houston.
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Presented in part at the Association for Research in Vision and Ophthalmology Annual Meeting, Sarasota, 1989, and as a poster at the American Academy of Ophthalmology Annual Meeting, New Orleans, OCl/Nov 1989. Supported by grants from the Deutsche Forschungsgemeinschaft (Ho 1111/1-1, 1·2), West Germany, the Retina Research Foundation, the Lions Eye Bank, Houston, Texas, and Research to Prevent Blindness, Inc, New York, New York. Reprint requests to Ramon L. Font, MD, Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
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Herpes simplex corneal disease is the leading infectious cause of unilateral blindness in the industrialized countries. 1 Its pathogenesis and optimal management remain controversial. Clinical and experimental studies have suggested that herpetic stromal keratitis is probably a result of viral infection and immunologic mechanisms although the exact roles of each have not been completely defined. 2 - 5 Granulomatous cell reactions of the cornea may constitute a common complication in stromal keratitis which may lead to scarring and perforation. If they are centered around Descemet's membrane, they may be recognized clinically.6 It has been suggested that the granulomatous reactions around Descemet's membrane are nonspecific as they have been observed in various conditions. 7,s Although corneal granulomatous cell reactions already have
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been reported in the first half of this century9 and later described in more detail,IO-14 their nature is still controversial. In two previous studies, herpes simplex virus (HSV) antigens were not found in the foci of granulomatous reactions at the level of Descemet's membrane. 15.16 Clinical observations,17,18 experimental studies, 19-22 HSV isolation 23 and its immunofluorescent demonstration in the anterior chamber4.25 suggested a role of the corneal endothelium in the pathogenesis of herpetic stromal keratitis and keratouveitis. To our knowledge, however, the presence of HSV in human corneal endothelium has not yet been documented. This article studies the distribution of granulomatous reactions in herpetic stromal keratitis and determines if herpes simplex antigens occur in human corneal endothelial cells as well as in the epithelioid cells of the granulomatous reactions centered around Descemet's membrane.
MATERIALS AND METHODS Two hundred fifteen corneal buttons were obtained by penetrating (207/215 or 96.3%) or lamellar keratoplasty (8/215 or 3.7%) from patients with a previous clinical diagnosis of herpes simplex stromal keratitis and a definite history of dendritic epithelial or recurrent herpetic stromal disease. Preoperatively, the patients presented with clinically inactive stromal scarring, nonulcerative or ulcerative necrotizing stromal keratitis. Inactive disease for at least 6 months was defined using clinical criteria (e.g., corneal scarring in a white and quiet eye without evidence of epithelial keratitis, recurrent stromal disease, anterior chamber reaction, or mild anterior uveitis). The keratectomy specimens were fixed in 10% buffered formalin. If localized lesions were present, sections were taken through the lesions in order to find areas that were representative of the pathologic changes noted or suspected from the clinical and gross examinations. Multiple sections, cut at 5 JLm from paraffin-embedded tissues, were available from each specimen. Hematoxylin-eosin and periodic acid-Schiff stains were routinely performed. Special stains including Brown and Hopps (modified Gram stain), Grocott's methenamine silver, and Ziehl-Neelsen for acid-fast bacilli were used when indicated. The sections were examined for the presence of granulomatous cell reactions at the level of Bowman's layer, the midstroma or Descemet's membrane. The distribution of the granulomatous reactions was analyzed according to their location centrally, paracentrally, or peripherally. Sections adjacent to those containing granulomatous reactions were deparaffinized by placing the slides in the oven at 51°C for 2 hours followed by immersion in xylene and hydration in decreasing ethanol concentrations. Endogenous peroxidase activity was neutralized using 100% methanol and 0.4% hydrogen peroxide. Nonimmunologic binding of antibodies was blocked by incubation with normal serum (Vectastain avidin-biotin complex [ABC], horse for anti-IgG, and goat for anti-IgM secondary antibodies). Incubation with all primary antibodies was car-
ried out in a moist chamber at 37°C for 60 minutes. The immunohistochemical stainings for HSV-1 antigens, UCHL-1 (mature activated T-cells, subpopulation of resting T-cells within CD4 and CD8 subsets), LN-2 (Blymphocytes), and Leu-7 (natural killer cells) antigens were carried out using commercially available versions of the avidin-biotinylated peroxidase method (Vectastain ABC, for rabbit and mouse IgG as well as mouse IgM [Vector Laboratories, Burlingame, CA]), as previously described. 26 Herpes simplex virus antigens were typed as specific to HSV -1 using monoclonal antibodies directed against epitopes on the ICP 4 and ICP 6 proteins27 of HSV-l (courtesy of Dr. H.B. Qavi, Department of Molecular Virology, Baylor College of Medicine, and Dr. M. Zweig, National Cancer Institute). Specificity and sensitivity of the viral antisera were determined by applying them to human tissues that were culture-proven positive for HSV-l. There was no cross-reactivity with cytomegalovirus antigens. Sections of human thymus and tonsils provided the sensitivity and specificity controls for the demonstration ofUCHL-1, LN-2, and Leu-7 antigens. In control series, the primary antibodies were omitted or substituted by normal rabbit serum or unrelated IgG and IgM mouse monoclonal antibodies. Negative tissue controls included keratectomy specimens with stromal dystrophies from patients without a history of ocular herpes simplex infection. False-positive reactions as seen on edges of the sections were excluded from evaluation. The primary antibodies to HSV -1 (Macintyre strain, lot c 77, 1: 600, ABC) and UCHL-1 (lot 038, 1:20) were from Dako, Santa Barbara, CA. The murine monoclonal anti-antibodies to Leu-7 (lot 0425, prediluted) and LN-2 (lot 8012 B, prediluted) were obtained from Becton Dickinson, Mountain View, CA, and Techniclone International, Santa Ana, CA. Horse anti-mouse IgG (HSV-1, UCHL-1, LN-2; ABC), goat anti-mouse IgM (Leu-7; ABC), and goat anti-rabbit IgG (HSV; ABC) were used as secondary antisera. The sections were incubated with these reagents in a moist chamber at 37°C for 30 minutes. Subsequent application of the avidin-biotinylated peroxidase complex (Vectastain) was carried out under the same conditions for 45 minutes. The sites of peroxidase activity were visualized by incubation offreshly prepared 3,3'-diaminobenzidine tetrahydrochloride (Fluka, Buchs, Switzerland), 5 mg in 10 ml of 0.05 M trishydroxymethylaminomethane buffered saline, pH 7.6, containing 0.0015 % hydrogen peroxide. The slides were counterstained with Gill's hematoxylin, dehydrated, cleared in xylene, and mounted in Histoclad medium (c. Adams, Parsippany, NJ). Statistical analyses for comparing data were performed with the chi-square test.
RESULTS CLINICOPATHOLOGIC CORRELATIONS
Fifty-three (25%) of 215 keratectomy specimens from patients with a clinical diagnosis of herpes simplex stromal
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Fig 1. Top left. central corneal ulcer with descemetocele and impending perforation in a 73-year-old patient who had a history of herpetic stromal keratitis for I y ear. Folds at the level of Descemet's membrane radiate superiorly from the center of the ulcer. Fig 2. Top right. paracentral view of section of the penetrating keratectomy specimen from the patient shown in Figure I. Herpes simplex viral antigens (brown reaction product) are detected in the deeply located stromal keratocytes adjacent to the ulcer with descemetocele (on the right. not shown in this field) (avidin-biotin complex [ABC] method hematoxylin counterstain ; original magnification, X64). Fig 3. Second row left. highpower view of the descemetocele of the same patient shown in Figure I illustrates thinning and splitting of Descemet's membrane with herpes simplex antigens (brown reaction product) on its anterior surface as well as in a small multinucleated giant cell (arrow) clinging to this membrane (ABC method, hematoxylin counterstain; original magnification. X 1(0). Fig 4. Second row right. keratectomy specimen from a 46-year-old man who had recurrent herpetic stromal disease for 15 years followed by a central corneal ulcer. This paracentral view (adjacent to the ulcer) displays deep stromal scarring with scattered neutrophils. Herpes simplex antigens are readily identified in the deep stromal keratocytes. Notice that the endothelial cells do not express detectable viral antigens (ABC method. hematoxylin counterstain ; original magnification , X I00). Fig 5. Third row left. keratectomy specimen from a 2 3-year-old woman who had recurrent herpetic stromal disease for 6 years followed by a central perforated corneal ulcer. Highpower view displays herpes simplex antigens (brown reaction product) in epithelioid
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keratitis and a history of dendritic or recurrent herpetic stromal keratitis displayed granulomatous cell reactions. The 53 patients included 35 males and 18 females (1.9: 1). Their ages at the time of surgery ranged from 10 to 88 years (median, 54 years). Two of 53 patients (3.8%) with granulomatous reactions had bilateral herpetic keratitis. The 53 corneal buttons were obtained exclusively by penetrating keratoplasty. Forty-five (85%) of the 53 patients had undergone a single keratoplasty, four had had two keratoplasties and two had been grafted three times. One patient had had a lamellar keratoplasty followed by a penetrating keratoplasty and two had had only a lamellar keratoplasty. Of the 53 keratectomy specimens, 22 were from patients with ulcerative necrotizing keratitis (Figs 1-6) and 31 from patients with stromal scarring or nonulcerative keratitis (Figs 7-10). Granulomatous reactions were identified significantly more often in specimens from patients with ulcerative necrotizing keratitis (22/38 or 58%; Figs 1-6) than in those with stromal scarring or nonulcerative keratitis (31/177 or 17%; Figs 7-10) (P < 0.00001). All 13 patients with ulcerative necrotizing keratitis who had had corneal perforation showed histopathologically a granulomatous reaction around Descemet's membrane (Fig 5). Corneal granulomatous reactions. Granulomatous reactions were identified histopathologically mostly in the deep stroma and around Descemet's membrane, centrally (50/53 or 94%; Figs 3,5, 10). In 25% (13/53), they were observed in the midstroma and in 9% (5/53) at the level of Bowman's layer. Fifteen keratectomy specimens (28%) displayed granulomatous reactions with simultaneous involvement of Descemet's membrane, the midstroma, and/or Bowman's layer. The granulomatous reactions were located mostly in the central cornea (66% or 35/53; Figs 3, 5, 10), less frequently in the paracentral region (21% or 11/53), or exclusively in the peripheral cornea (13% or 7/53). Lymphocytes that were entrapped in the granulomatous cell foci stained positively for UCHL-1, but negatively for LN-2 and Leu-7 antigens. CLINICO-IMMUNOPATHOLOGIC CORRELAnONS
Immunocytochemical studies of HSV antigens were performed on sections from 58 corneal buttons that had been obtained by penetrating keratoplasty.
Herpes simplex virus antigens were identified significantly more often in keratectomy specimens from patients with ulcerative necrotizing keratitis (91 % or 20/22; Figs 1-6) than in those with non ulcerative nonnecrotizing stromal keratitis and stromal scarring (11 % or 4/36; Figs 7-10) (P < 0.005). Herpes simplex virus antigens were demonstrated more commonly in avascular (92% or 22/24) than in vascularized corneal lesions (8% or 2/24) (P < 0.005). Ulcerative necrotizing stromal keratitis. Twenty (91 %) of 22 keratectomy specimens from patients with ulcerative necrotizing stromal keratitis displayed HSV antigens. All the patients had a deep stromal ulcer at the time of surgery (Fig 1). Thirteen (59%) of the 22 patients presented with corneal perforation. Herpes simplex virus antigens were found mostly in stromal keratocytes and the extracellular stroma (20/20 or 100%; Figs 2, 4), less commonly in endothelial cells (3/20 or 15%; Fig 6), epithelial cells (3/20 or 15%), or epithelioid histiocytes and multinucleated giant cells (3/20 or 15%; Figs 3, 5). Herpes simplex virus antigens were identified mostly within the central portion of the corneal buttons (15/20 or 75%), and less frequently in the peripheral third (3/20 or 15%) or in both central and peripheral portions (2/20 or 10%). The antigens were distributed more often in the deep corneal stroma (14/20; Figs 2, 4)-particularly along Descemet's membrane and the endothelial cells-than in the superficial stroma (2/20) or in both (4/20). Nonulcerative nonnecrotizing stromal keratitis and disciform stromal scarring. Four (11 %) of the 36 keratectomy specimens with non ulcerative nonnecrotizing stromal keratitis or disciform stromal scarring showed HSV antigens. Preoperatively, these four patients had predominantly an avascular scar and thinned stroma centrally (Fig 7). By slit-lamp examination, irregularities at the level of Descemet's membrane (Fig 7) were noted in two patients. The latter were found to correspond histopathologically to granulomatous reactions at the level of Descemet's membrane. Clinically, there was no evidence of epithelial or stromal keratitis or anterior chamber reaction. None of these patients had a recurrence during the last 6 months. Two patients were without any treatment during that time;
(
histiocytes as well as small multinucleated giant cells located at the level of the artifactually detached Descemet's membrane. A focal break in Descemet's membrane is readily observed (ABC method, hematoxylin counterstain; original magnification, X 100). Fig 6. Third row right, highpower view of area located more peripherally to that seen in Figure 5. Herpes simplex viral antigens are detected in corneal endothelial cells. The stromal keratocytes do not stain for herpes simplex antigens (ABC method, hematoxylin counterstain; original magnification, X 100). Fig 7. Fourth row left, left cornea of a 10-year-old girl with a central disciform scar with thinning of the stroma. She had a history of herpetic stromal keratitis for 6 years, with no evidence of recurrence during the last 16 months. Notice the opacities at the level of Bowman's layer and the irregularities at the level ofthe endothelium. Visual acuity was 20/100. Fig 8. Fourth row right, paracentral view of section of the corneal button obtained by penetrating keratoplasty from the patient shown in Figure 7. The corneal epithelium is moderately acanthotic. Bowman's layer is focally interrupted. The adjacent superficial stroma shows numerous plump histiocytes containing periodic acid-Schiff (PAS)-positive granules. The stroma centrally is thinned and scarred (on the right) (PAS; original magnification, X40). Fig 9. Bottom left, section of cornea from the same patient shown in Figure 7 displays stromal scarring. Herpes simplex viral antigens are identified exclusively in endothelial cells adjacent to a focus of granulomatous reaction at the level of Descemet's membrane (not seen in this field). Notice that the stromal keratocytes in the overlying scarred stroma do not express detectable viral antigens (ABC method, hematoxylin counterstain; original magnification, X 160). Fig 10. Bottom right, keratectomy specimen from a 29-yearold man with disciform stromal scarring. He had recurrent herpetic stromal keratitis for II years. There was no evidence of recurrence during the last 8 months. There is a florid granulomatous reaction with focal rupture of Descemet's membrane (PAS; original magnification, X 100).
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one of them had topical low-dose steroids (once daily) and acyclovir ointment (once daily), which was discontinued 2 months before surgery, and the fourth patient had topical low-dose steroids (once daily) until 3 months before surgery. Histopathologically, the four HSV-I-positive keratectomy specimens displayed central stromal scarring (Fig 8) with granulomatous foci around Descemet's membrane as well as in the stroma. Herpes simplex virus type 1 antigens were identified in corneal endothelial cells (3/4; Fig 9) and in epithelioid histiocytes and multinucleated giant cells 0/4). The HSVpositive endothelial cells were located adjacent to the foci of granulomatous cell reaction at the level of Descemet's membrane.
DISCUSSION In this clinicopathologic study of 215 keratectomy specimens from patients with herpes simplex keratitis, the presence of HSV antigens was demonstrated in corneal endothelial cells, epithelioid histiocytes and multinucleated giant cells located at the level of Descemet's membrane. To the best of our knowledge, this observation has not been documented previously. Of clinical interest is the expression of HSV antigens in all corneal cell types including endothelial cells. Herpetic epithelial keratitis is known to result from replicating HSV in epithelial cells.28,29 Herpes virus particles and antigens have been identified in stromal keratocytes in both clinically active and inactive disease. 30- 33 The immunocytochemical identification ofHSV antigens in corneal endothelial cells is consistent with experimental evidence showing that HSV replicated in cultured rabbit corneal endothelial cells. 22 Herpes simplex virus antigens also have been demonstrated in rabbit corneal endothelial cells after topical and intracameral application of different strains of HSV. 19,20 In our study, HSV antigens were detected more often in keratocytes than in endothelial cells. These observations parallel the results of experimental studies showing that cultures of rabbit keratocytes in vitro produce higher amounts of infectious virus than those of rabbit endothelial cells. 22 Clinical observations correlating the area of endothelial cell damage to the area of retrocorneal precipitates in herpetic keratouveitits l7 ,18 provided indirect evidence suggesting that involvement of the corneal endothelium may play a significant role in ocular herpetic disease. Herpes simplex virus isolation from the anterior chamber has been documented in patients with anterior uveitis lacking active epithelial or stromal keratitis.23 Our study clearly documents direct involvement of the corneal endothelium in both herpetic disciform stromal scarring and ulcerative necrotizing keratitis. Our results lend support to clinical observations suggesting the promising role of systemic antiviral treatment in deep herpetic keratitis. 34- 36 Since a randomized, controlled, double-masked clinical trial to evaluate the efficacy of oral 726
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acyclovir has not yet been performed, further clinical studies are needed on the judicious administration of topical and systemic antiviral agents in the treatment of herpes simplex stromal and endothelial keratitis. The demonstration of HSV antigens in deep stromal keratocytes and endothelial cells shows clearly that these cells can be targets of the HSV. Furthermore, this provides additional evidence in favor of performing a penetrating corneal graft over a lamellar keratoplasty in the surgical management of herpes simplex corneal disease. Granulomatous reactions of the cornea (Descemet's membrane, stroma and Bowman's layer) were shown to constitute a common tissue response in herpes simplex stromal keratitis, both in the necrotizing and nonnecrotizing types. Their real incidence is probably even higher since we did not routinely perform serial sections from every specimen. The distribution pattern for HSV antigens and the location of the foci of granulomatous reactions was similar in the keratectomy specimens studied. Both occurred throughout all corneal layers involving predominantly the central, deep stroma, adjacent to Descemet's membrane and the neighboring endothelial cells. Their common location in the deep stroma may be related to the stage of the disease at the time of grafting and the previous topical application of antiviral agents and/or steroids with insufficient penetration and concentration of these drugs at the site of activity. All 13 patients in our study who presented with ulcerative necrotizing herpes simplex keratitis and perforation showed histopathologically HSV-associated granulomatous reactions centered around Descemet's membrane. The formation of a granulomatous reaction around Descemet's membrane often precedes corneal perforation in ulcerative necrotizing keratitis. 6 In this situation, its clinical recognition 6,37 may represent an indication for penetrating keratoplasty. The granulomatous host tissue response in herpes simplex keratitis may be regulated by specific nucleotide sequences of HSV 38 or represent a characteristic tissue response regarding the immunologic status of the patient. Possible targets of the granulomatous reactions in herpes simplex corneal disease include the presence of viral antigens as well as cellular and/or basement membrane structures from the host (e.g., Descemet's membrane) which may become antigenically altered by the virus or the disease process, or both. Our findings suggest that a T-cell-mediated immunity plays a major role in the pathogenesis of granulomatous reactions in herpes simplex keratitis. The identification of HSV antigens in the stromal keratocytes and adjacent granulomatous foci of keratectomy specimens from patients who preoperatively had clinically inactive stromal scarring is consistent with results of in vitro cultural isolation of HSV from patients with clinically inactive herpetic stromal scarring. 39-4 I Previous reports42 and our current findings suggest that the clinical impression of "inactive" stromal scarring is not completely reliable, since histopathologic examination ofkeratectomy specimens from these cases demonstrates lymphocytic infiltrates and/or granulomatous foci associated with the presence of HSV antigens. The virus may persist
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within some cells in a slowly replicating form as a lowgrade chronic infection and provoke a mitigated host tissue response. Animal models have provided evidence of a persistent/latent type of infection in peripheral nonneural tissues (skin and cornea).43-46 The presence of HSV antigens and granulomatous reactions was significantly correlated with the necrotizing type of herpetic stromal keratitis. This important clinicopathologic correlation as well as the immunocytochemical identification ofHSV antigens in endothelial cells ofkeratectomy specimens from patients with both ulcerative necrotizing and nonnecrotizing disciform stromal keratitis may provide a further basis for the diagnosis and management of herpes simplex stromal and endothelial disease.
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simplex virus type 1 gene that causes cell fusion. Virology 1985: 145:
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169:91-6. 44. Cook SD, Brown SM. Herpes simplex virus type 1 persistence and latency in cultured rabbit comeal epithelial cells, keratocytes, and endothelial cells. Br J Ophthalmol 1986; 70:642-50. 45. Sabbaga EMH, Pavan-Langston D, Bean KM, Dunkel EC. Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease. Exp Eye Res 1988; 47:545-53. 46. O'Brien WJ, Taylor JL. The isolation of herpes simplex virus from rabbit comeas during latency. Invest Ophthalmol Vis Sci 1989; 30:
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