HERPETIC STROMAL KERATITIS—EVIDENCE FOR CELL-MEDIATED IMMUNOPATHOGENESIS JOSEPH F. METCALF, P H . D . , AND HERBERT E. KAUFMAN, M.D. Gainesville, Florida
Although herpes simplex virus can pen etrate the corneal epithelium and invade the stroma, it has not been possible to explain the pathology of stromal herpes solely on the basis of viral infection and death of keratocytes.1,2 Despite recent ad vances in understanding and classifying the clinical phases of herpetic stromal keratitis, and advances in therapy with antiviral agents and corticosteroids, the treatment of this deep stromal disease is unsatisfactory.3 We estimate that at least 10% of the corneal transplants at Shand's Teaching Hospital are performed because of stro mal disease caused by herpes simplex virus. These patients have an extended history of recurrent infection that re sponds temporarily to standard therapeu tic procedures. Although the corneal transplant is successful in restoring sight to persons blinded by herpetic stromal disease, these cases are medical failures because of the present lack of knowledge regarding the cause and pathogenesis of stromal keratitis of herpetic origin.1,4 Delayed hypersensitivity, or cellmediated immunity, may be involved in the pathogenesis of disciform edema and stromal keratitis. Williams, Nesburn, and Kaufman5 found that prior subcutaneous inoculation of rabbits with live herpes From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida. This study was supported by grants EY01580 and EY-00007 from the National Eye Insti tute. Presented at the Spring Meeting of the Associa tion for Research in Vision and Ophthalmology, April 26-30, 1976, Sarasota, Florida. Reprint requests to J. F. Metcalf, Ph.D., Depart ment of Ophthalmology, College of Medicine, Box J-184, J. Hillis Miller Health Center, Gainesville, FL 32610.
simplex virus resulted in a significant increase in the incidence of experimental disciform keratitis as compared with ani mals that had not been presensitized. The average number of days to onset of the disease (73/4 days) was less in animals receiving subcutaneous inoculation than in controls (nine days). Swyers, Lausch, and Kaufman6 sensi tized guinea pigs by intradermal inocula tion of viable herpes virus (strain HE 17) and later challenged the animals with an intracorneal injection of soluble viral an tigen. Biomicroscopic and histologie ob servations of the cornea showed a close correlation between the development of corneal edema and the infiltration of lym phocytes that was reached 72 hours after challenge. This observation is consistent with previous studies7 showing a delayed hypersensitivity to herpes simplex virus antigens in guinea pig skin that could be passively transferred with cells but not with serum. Recently Henson and associates8 dem onstrated the presence of herpes simplex virus antigens on the surface membranes of productively infected corneal (SIRC) cells in vitro. They suggested that stromal keratitis is analogous to a host-vs-graft reaction in which individual infected cells are recognized as foreign by the host organism because of the presence of her pes simplex virus neoantigens on the cell surface, thus initiating rejection of the stromal cells and resulting in tissue ne crosis and destruction of collagen. Finally, like many types of hypersensi tivity, disciform edema in humans re sponds well to small doses of topical corticosteroids . 9 · 1 0 Previous studies 11 have shown that rab bit corneas with experimentally induced
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herpetic stromal keratitis are infiltrated with polymorphonuclear leukocytes, plas ma cells, macrophages, and lymphocytes. These observations indicate that com plex immunological events are associ ated with the inflammatory process. In this report we describe ultrastructural and immunofluorescence studies in rabbit and human corneas which suggest that a cell-mediated immune response is also involved in the pathogenesis of this blinding disease. M A T E R I A L AND M E T H O D S
Animal model—An animal model of herpetic stromal keratitis with a clinical syndrome similar to the disease observed in human patients, including collagen necrosis and vascularization of the cor nea, has recently been described. 1 1 The corneas of albino rabbits were injected intrastromally with 0.02 ml of a suspen sion of the RE strain of herpes simplex virus. Central stromal edema and opacification appeared within five to seven days and the inflammatory response reached maximum intensity usually within 14 to 18 days. The disease resolved in three to four weeks except in animals that devel oped necrotizing keratitis. Human corneas—Corneal buttons were obtained from eight patients who re ceived penetrating keratoplasties for her petic stromal keratitis at Shands Teaching Hospital. These individuals had a history of recurrent herpetic keratitis and stromal necrosis that failed to respond to treat ment with antiviral agents and corticosteroids. Fluorescence microscopy—Serum· was obtained from an individual with a high titer of antiherpes simplex virus neutra lizing antibody (1:1024 serum dilution) as determined by the 50% endpoint method, using tube cultures of human embryonic kidney cells. Immunoglobulin G (IgG) was isolated by sodium sulfate fractionation 12 and conjugated with fluorescein
DECEMBER, 1976
isothiocyanate (FITC). 13 - 14 Specificity of the F I T C conjugate was demonstrated by blocking procedures with specific unlabeled antigen and lack of staining with control tissues. Rabbit corneas were quick-frozen in powdered dry ice. Cryostat sections (8 μ) were mounted on gelatine-coated slides and air dried. The sections were incubat ed with the fluorescein-labeled antibody in a moist chamber for one hour at room temperature, then rinsed in tris-saline by shaking for ten minutes. Cover slips were mounted with carbonate-buffered glycerol. The sections were examined with a Zeiss fluorescence microscope, and pho tographed (Polaroid 107 film) using an exposure time of three minutes. Electron microscopy—Corneas were fixed overnight in cold, phosphate-buf fered glutaraldehyde (2.5%), postfixed for one hour in 1% osmium, and embed ded in Epon. Thin sections were stained with lead citrate and uranyl acetate, and examined with a Zeiss EM-9 electron microscope. Viral cultures—Tube cultures of human embryonic kidney cells were seeded with macerated pieces of cornea. The cultures were examined at three and six days for evidence of viral infection. RESULTS
Rabbit corneas—Histological and elec tron microscopic examination of rabbit corneas from experimental animals with either disciform edema or early necrotiz ing keratitis (14 to 21 days after viral inoculation) revealed the presence of nu merous lymphocytes, plasma cells, mac rophages, and polymorphonuclear leuko cytes. 11 Lymphocytes were frequently found in close contact with keratocytes in various stages of degeneration (Fig. 1). Degenerating keratocytes showed disrup tion of the endoplasmic reticulum with vesicles and electron-dense granular ma terial in the cytoplasm. Viral particles
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Fig. 1 (Metcalf and Kaufman). Lymphocyte (L) is in close contact with cytoplasmic process of degenerating keratocyte (arrow) in rabbit cornea with disciform edema. Bar gauge = 1 μ.
were not found in any of the degenerating keratocytes by electron microscopic ex amination. Although free lymphocytes, not associated with other cells, were fre quently seen, none were observed in close contact with cells other than keratocytes in the inflamed corneas. Free lympho cytes were round or oval and had indent ed nuclei with a few mitochondria and many polyribosomes in the cytoplasm. Microvilli were prominent on the cell surface. Lymphocytes in direct contact with keratocytes assumed a more flat tened or elongated profile and microvilli were not evident. Immunofluorescent labeling of cryostat sections of rabbit corneas obtained from animals with disciform edema showed
viral antigen in keratocytes of the anterior stroma (Fig. 2), and in keratocytes and inflammatory cells located in foci deep in the stroma (Fig. 3). Viral antigens were not found in the epithelium at this stage of the experimentally induced disease, as shown by the absence of fluorescence (Fig. 3). Viral cultures of rabbit corneas were positive up to 14 days after corneal inocu lation, during the active stage of infec tion, but all cultures were negative after the 14th day. However, immunofluorescence showed viral antigen in these in flamed tissues with early necrotizing keratitis. Human corneas—Electron microscopic examination of corneal buttons obtained
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Fig. 2 (Metcalf and Kaufman). Immunofluorescent localization of herpes simplex virus antigen in keratocytes of the anterior stroma of a rabbit cornea with disciform edema. Note the absence of viral antigen in the epithelium. Bar gauge = 10 μ.
from eight patients after keratoplasty for herpetic stromal disease showed that typ ical lymphocytes were present in these corneas also (Fig. 4). Lymphocytes were frequently found in direct contact with degenerating keratocytes (Figs. 5 and 6). Viral particles were found in only two of the eight human corneas examined by electron microscopy. However, viral par ticles were not found in the degenerated keratocytes that had lymphocytes in close contact, and attempts to demonstrate the presence of virus by tissue culture tech niques were negative. One lymphocyte (Fig. 6) appeared to have specialized cytoplasmic processes directed toward the keratocyte.
DISCUSSION
Herpes simplex virus induces a cellmediated response to subcutaneous infec tion, 7,15 and cell-mediated immunity has been implicated as a possible cause of experimental disciform edema of the cor nea. 5 , 6 In the present study of herpetic stromal keratitis, fluorescence microscopy showed viral antigens in the keratocytes of the cornea, although electron micro scopic observations and viral cultures show that mature infectious viral parti cles may be absent from the same tissues. A major infiltrating cell type found in the corneas of experimental animals and hu mans with stromal keratitis is the lym phocyte, many of which are found in
Fig. 3 (Metcalf and Kaufman). Localization of viral antigen in keratocytes and inflammatory cells in the midstroma of rabbit cornea. Bar gauge = 20 μ.
Fig. 4 (Metcalf and Kaufman). Lymphocyte in the stroma of human cornea. The stromal collagen fibers surrounding the cell are sparse and disrupted. Bar gauge = 2 μ.
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Fig. 5 (Metcalf and Kaufman). Lymphocyte (L) in human cornea is adjacent to degenerated processes of two keratocytes (arrows). Bar gauge = 2 μ.
intimate contact with keratocytes in vary ing degrees of degeneration. Electron microscopic studies of reject ing corneal homografts16'17 show that cell damage is confined to areas of lymphocytic invasion, and the presence of lympho cytes enveloping keratocytes and lym phocytes with cytoplasmic processes di rected toward degenerating endothelial cells and Descemet's membrane was demonstrated. Although the precise mechanism by which immunological pathology occurs is unknown, the presence of viral antigens in stromal keratocytes and the association of lymphocytes with degenerating kerato cytes and stromal collagen fibers suggest that a mechanism similar to the one pro
posed for homograft rejection18 may oper ate in the corneas of rabbits and humans with herpetic stromal keratitis. The Tlymphocytes are probably responsible for cell-mediated immune reactions. Specifi cally sensitized T-lymphocytes possess the ability to recognize and destroy cells bearing specific cell surface antigens. When viral particles invade a living cell, either productive infection involving viral replication and cell destruction, or abortive infection in which viral replica tion is blocked, may occur.19,20 A fraction of the abortively infected cells may pro duce viral antigens, however, that interact with lymphocytes and induce blast trans formation leading to the production of many lymphocytes specifically sensitized
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Fig. 6 (Metcalf and Kaufman). Lymphocyte (L) in close proximity to the cytoplasmic process of a keratocyte (arrow) in human cornea. Bar gauge = 1 μ.
to the viral antigen. These lymphocytes then enter the infected tissue and destroy the antigenic cells. Our observations are consistent with this model of cell-mediated immunopathogenesis in experimental stromal keratitis in the rabbit, and in herpetic stromal keratitis of human patients also. SUMMARY
Immunofluorescence, histological, and electron microscopic observations were made on rabbit corneas from animals with experimentally induced stromal ker atitis following intracorneal injection with the RE strain of herpes simplex virus. Electron microscopic observations were also made on human corneas ob
tained from patients with a history of herpetic stromal disease. Viral antigens were demonstrated by immunofluores cence in keratocytes of rabbit corneas with herpetic stromal keratitis. Electron microscopic observations and viral cul ture failed to reveal the presence of viral particles in these tissues. Lymphocytes, a major infiltrating cell type found in both the rabbit and human corneas, were often found in intimate contact with degenerat ing keratocytes. REFERENCES 1. Pettit, T. H.: Herpes simplex keratitis: current concepts. In Becker, B., and Bürde, R. M. (eds.): Current Concepts in Ophthalmology. St. Louis, C. V. Mosby, 1969, vol. 2, p. 10.
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2. Kaufman, H. E.: Herpetic stromal disease, edi torial. Am. J. Ophthalmol. 80:1092, 1975. 3. : Systemic therapy of ocular herpes. Int. Ophthalmol. Clin. 15:163, 1975. 4. Laibson, P. R.: Corneal transplantation in the treatment of active and inactive herpes simplex keratitis. In Golden, B. (ed.): Ocular Inflammatory Disease. Springfield, Charles C Thomas, 1974, p. 297. 5. Williams, L. E., Nesburn, A. B., and Kaufman, H. E.: Experimental induction of disciform kerati tis. Arch. Ophthalmol. 73:112, 1965. 6. Swyers, J. S., Lausch, R. N., and Kaufman, H. E.: Corneal hypersensitivity to herpes simplex. Br. J. Ophthalmol. 51:843, 1967. 7. Lausch, R. N., Swyers, J. S., and Kaufman, H. E.: Delayed hypersensitivity to herpes simplex virus in the guinea pig. J. Immunol. 96:981, 1966. 8. Henson, D., Heimsen, R., Becker, K. E., Strano, A. J., Sullivan, M., and Harris, D.: Ultrastructural localization of herpes simplex virus antigens on rabbit corneal cells using sheep antihuman IgG antihorse ferritin hybrid antibodies. Invest. Oph thalmol. 13:819,1974. 9. Kaufman, H. E.: Therapy of corneal virus dis ease. In King, J. H., and McTigue, J. W. (eds.): The Cornea—World Congress. Washington, 1965, p. 187. 10. : Treatment of deep herpetic keratitis with IDU and corticosteroids. Eye Ear Nose Throat Digest. 25:37, 1963. 11. Metcalf, J. F., McNeill, J. I., and Kaufman, H. E.: Experimental disciform edema and necrotizing
DECEMBER, 1976
keratitis in the rabbit. Invest. Ophthalmol. In press. 12. Breese, S. S., Jr., and Hsu, K. D.: Techniques of ferritin-tagged antibodies. In Maramorosch, K., and Koprowski, H. (eds.): Methods in Virology, vol. 5. New York, Academic Press, 1971, p. 404. 13. Weir, D. M.: Handbook of Experimental Im munology. Oxford, Blackwell Scientific Publica tions, 1967, p. 581. 14. Goldman, M.: Fluorescent Antibody Meth ods. New York, Academic Press, 1968, p. 103. 15. Oakes, J. E.: Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection. Infect. Immun. 12:166, 1975. 16. Kanai, A., and Polack, F. M.: Ultramicroscopic changes in the corneal graft stroma during early rejection. Invest. Ophthalmol. 10:415, 1971. 17. Polack, F. M., and Kanai, A.: Electron micro scopic studies of graft endothelium in corneal graft rejection. Am. J. Ophthalmol. 73:711, 1972. 18. Gowars, J. L.: Immunology of the small lym phocyte. In Good, R. A., and Fisher, D. W. (eds.): Immunobiology. Stamford, Sinauer Associates, Inc., 1972, p. 18. 19. Kelleher, J. J., Varani, J., and Nelson, W. W.: Establishment of a nonproductive herpes simplex virus infection in rabbit kidney cells. Infect. Im mun. 12:128, 1975. 20. Green, M.: Oncogenic viruses. In Snell, E. E., Boyer, P. D., Meister, A., and Sinsheimer, R. L. (eds.): Annual Review of Biochemistry. Palo Alto, Annual Reviews, Inc., 1970, p. 701.
Although herpes simplex virus can pen etrate the corneal epithelium and invade the stroma, it has not been possible to explain the pathology of stromal herpes solely on the basis of viral infection and death of keratocytes.1,2 Despite recent ad vances in understanding and classifying the clinical phases of herpetic stromal keratitis, and advances in therapy with antiviral agents and corticosteroids, the treatment of this deep stromal disease is unsatisfactory.3 We estimate that at least 10% of the corneal transplants at Shand's Teaching Hospital are performed because of stro mal disease caused by herpes simplex virus. These patients have an extended history of recurrent infection that re sponds temporarily to standard therapeu tic procedures. Although the corneal transplant is successful in restoring sight to persons blinded by herpetic stromal disease, these cases are medical failures because of the present lack of knowledge regarding the cause and pathogenesis of stromal keratitis of herpetic origin.1,4 Delayed hypersensitivity, or cellmediated immunity, may be involved in the pathogenesis of disciform edema and stromal keratitis. Williams, Nesburn, and Kaufman5 found that prior subcutaneous inoculation of rabbits with live herpes From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida. This study was supported by grants EY01580 and EY-00007 from the National Eye Insti tute. Presented at the Spring Meeting of the Associa tion for Research in Vision and Ophthalmology, April 26-30, 1976, Sarasota, Florida. Reprint requests to J. F. Metcalf, Ph.D., Depart ment of Ophthalmology, College of Medicine, Box J-184, J. Hillis Miller Health Center, Gainesville, FL 32610.
simplex virus resulted in a significant increase in the incidence of experimental disciform keratitis as compared with ani mals that had not been presensitized. The average number of days to onset of the disease (73/4 days) was less in animals receiving subcutaneous inoculation than in controls (nine days). Swyers, Lausch, and Kaufman6 sensi tized guinea pigs by intradermal inocula tion of viable herpes virus (strain HE 17) and later challenged the animals with an intracorneal injection of soluble viral an tigen. Biomicroscopic and histologie ob servations of the cornea showed a close correlation between the development of corneal edema and the infiltration of lym phocytes that was reached 72 hours after challenge. This observation is consistent with previous studies7 showing a delayed hypersensitivity to herpes simplex virus antigens in guinea pig skin that could be passively transferred with cells but not with serum. Recently Henson and associates8 dem onstrated the presence of herpes simplex virus antigens on the surface membranes of productively infected corneal (SIRC) cells in vitro. They suggested that stromal keratitis is analogous to a host-vs-graft reaction in which individual infected cells are recognized as foreign by the host organism because of the presence of her pes simplex virus neoantigens on the cell surface, thus initiating rejection of the stromal cells and resulting in tissue ne crosis and destruction of collagen. Finally, like many types of hypersensi tivity, disciform edema in humans re sponds well to small doses of topical corticosteroids . 9 · 1 0 Previous studies 11 have shown that rab bit corneas with experimentally induced
827
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herpetic stromal keratitis are infiltrated with polymorphonuclear leukocytes, plas ma cells, macrophages, and lymphocytes. These observations indicate that com plex immunological events are associ ated with the inflammatory process. In this report we describe ultrastructural and immunofluorescence studies in rabbit and human corneas which suggest that a cell-mediated immune response is also involved in the pathogenesis of this blinding disease. M A T E R I A L AND M E T H O D S
Animal model—An animal model of herpetic stromal keratitis with a clinical syndrome similar to the disease observed in human patients, including collagen necrosis and vascularization of the cor nea, has recently been described. 1 1 The corneas of albino rabbits were injected intrastromally with 0.02 ml of a suspen sion of the RE strain of herpes simplex virus. Central stromal edema and opacification appeared within five to seven days and the inflammatory response reached maximum intensity usually within 14 to 18 days. The disease resolved in three to four weeks except in animals that devel oped necrotizing keratitis. Human corneas—Corneal buttons were obtained from eight patients who re ceived penetrating keratoplasties for her petic stromal keratitis at Shands Teaching Hospital. These individuals had a history of recurrent herpetic keratitis and stromal necrosis that failed to respond to treat ment with antiviral agents and corticosteroids. Fluorescence microscopy—Serum· was obtained from an individual with a high titer of antiherpes simplex virus neutra lizing antibody (1:1024 serum dilution) as determined by the 50% endpoint method, using tube cultures of human embryonic kidney cells. Immunoglobulin G (IgG) was isolated by sodium sulfate fractionation 12 and conjugated with fluorescein
DECEMBER, 1976
isothiocyanate (FITC). 13 - 14 Specificity of the F I T C conjugate was demonstrated by blocking procedures with specific unlabeled antigen and lack of staining with control tissues. Rabbit corneas were quick-frozen in powdered dry ice. Cryostat sections (8 μ) were mounted on gelatine-coated slides and air dried. The sections were incubat ed with the fluorescein-labeled antibody in a moist chamber for one hour at room temperature, then rinsed in tris-saline by shaking for ten minutes. Cover slips were mounted with carbonate-buffered glycerol. The sections were examined with a Zeiss fluorescence microscope, and pho tographed (Polaroid 107 film) using an exposure time of three minutes. Electron microscopy—Corneas were fixed overnight in cold, phosphate-buf fered glutaraldehyde (2.5%), postfixed for one hour in 1% osmium, and embed ded in Epon. Thin sections were stained with lead citrate and uranyl acetate, and examined with a Zeiss EM-9 electron microscope. Viral cultures—Tube cultures of human embryonic kidney cells were seeded with macerated pieces of cornea. The cultures were examined at three and six days for evidence of viral infection. RESULTS
Rabbit corneas—Histological and elec tron microscopic examination of rabbit corneas from experimental animals with either disciform edema or early necrotiz ing keratitis (14 to 21 days after viral inoculation) revealed the presence of nu merous lymphocytes, plasma cells, mac rophages, and polymorphonuclear leuko cytes. 11 Lymphocytes were frequently found in close contact with keratocytes in various stages of degeneration (Fig. 1). Degenerating keratocytes showed disrup tion of the endoplasmic reticulum with vesicles and electron-dense granular ma terial in the cytoplasm. Viral particles
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Fig. 1 (Metcalf and Kaufman). Lymphocyte (L) is in close contact with cytoplasmic process of degenerating keratocyte (arrow) in rabbit cornea with disciform edema. Bar gauge = 1 μ.
were not found in any of the degenerating keratocytes by electron microscopic ex amination. Although free lymphocytes, not associated with other cells, were fre quently seen, none were observed in close contact with cells other than keratocytes in the inflamed corneas. Free lympho cytes were round or oval and had indent ed nuclei with a few mitochondria and many polyribosomes in the cytoplasm. Microvilli were prominent on the cell surface. Lymphocytes in direct contact with keratocytes assumed a more flat tened or elongated profile and microvilli were not evident. Immunofluorescent labeling of cryostat sections of rabbit corneas obtained from animals with disciform edema showed
viral antigen in keratocytes of the anterior stroma (Fig. 2), and in keratocytes and inflammatory cells located in foci deep in the stroma (Fig. 3). Viral antigens were not found in the epithelium at this stage of the experimentally induced disease, as shown by the absence of fluorescence (Fig. 3). Viral cultures of rabbit corneas were positive up to 14 days after corneal inocu lation, during the active stage of infec tion, but all cultures were negative after the 14th day. However, immunofluorescence showed viral antigen in these in flamed tissues with early necrotizing keratitis. Human corneas—Electron microscopic examination of corneal buttons obtained
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Fig. 2 (Metcalf and Kaufman). Immunofluorescent localization of herpes simplex virus antigen in keratocytes of the anterior stroma of a rabbit cornea with disciform edema. Note the absence of viral antigen in the epithelium. Bar gauge = 10 μ.
from eight patients after keratoplasty for herpetic stromal disease showed that typ ical lymphocytes were present in these corneas also (Fig. 4). Lymphocytes were frequently found in direct contact with degenerating keratocytes (Figs. 5 and 6). Viral particles were found in only two of the eight human corneas examined by electron microscopy. However, viral par ticles were not found in the degenerated keratocytes that had lymphocytes in close contact, and attempts to demonstrate the presence of virus by tissue culture tech niques were negative. One lymphocyte (Fig. 6) appeared to have specialized cytoplasmic processes directed toward the keratocyte.
DISCUSSION
Herpes simplex virus induces a cellmediated response to subcutaneous infec tion, 7,15 and cell-mediated immunity has been implicated as a possible cause of experimental disciform edema of the cor nea. 5 , 6 In the present study of herpetic stromal keratitis, fluorescence microscopy showed viral antigens in the keratocytes of the cornea, although electron micro scopic observations and viral cultures show that mature infectious viral parti cles may be absent from the same tissues. A major infiltrating cell type found in the corneas of experimental animals and hu mans with stromal keratitis is the lym phocyte, many of which are found in
Fig. 3 (Metcalf and Kaufman). Localization of viral antigen in keratocytes and inflammatory cells in the midstroma of rabbit cornea. Bar gauge = 20 μ.
Fig. 4 (Metcalf and Kaufman). Lymphocyte in the stroma of human cornea. The stromal collagen fibers surrounding the cell are sparse and disrupted. Bar gauge = 2 μ.
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Fig. 5 (Metcalf and Kaufman). Lymphocyte (L) in human cornea is adjacent to degenerated processes of two keratocytes (arrows). Bar gauge = 2 μ.
intimate contact with keratocytes in vary ing degrees of degeneration. Electron microscopic studies of reject ing corneal homografts16'17 show that cell damage is confined to areas of lymphocytic invasion, and the presence of lympho cytes enveloping keratocytes and lym phocytes with cytoplasmic processes di rected toward degenerating endothelial cells and Descemet's membrane was demonstrated. Although the precise mechanism by which immunological pathology occurs is unknown, the presence of viral antigens in stromal keratocytes and the association of lymphocytes with degenerating kerato cytes and stromal collagen fibers suggest that a mechanism similar to the one pro
posed for homograft rejection18 may oper ate in the corneas of rabbits and humans with herpetic stromal keratitis. The Tlymphocytes are probably responsible for cell-mediated immune reactions. Specifi cally sensitized T-lymphocytes possess the ability to recognize and destroy cells bearing specific cell surface antigens. When viral particles invade a living cell, either productive infection involving viral replication and cell destruction, or abortive infection in which viral replica tion is blocked, may occur.19,20 A fraction of the abortively infected cells may pro duce viral antigens, however, that interact with lymphocytes and induce blast trans formation leading to the production of many lymphocytes specifically sensitized
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Fig. 6 (Metcalf and Kaufman). Lymphocyte (L) in close proximity to the cytoplasmic process of a keratocyte (arrow) in human cornea. Bar gauge = 1 μ.
to the viral antigen. These lymphocytes then enter the infected tissue and destroy the antigenic cells. Our observations are consistent with this model of cell-mediated immunopathogenesis in experimental stromal keratitis in the rabbit, and in herpetic stromal keratitis of human patients also. SUMMARY
Immunofluorescence, histological, and electron microscopic observations were made on rabbit corneas from animals with experimentally induced stromal ker atitis following intracorneal injection with the RE strain of herpes simplex virus. Electron microscopic observations were also made on human corneas ob
tained from patients with a history of herpetic stromal disease. Viral antigens were demonstrated by immunofluores cence in keratocytes of rabbit corneas with herpetic stromal keratitis. Electron microscopic observations and viral cul ture failed to reveal the presence of viral particles in these tissues. Lymphocytes, a major infiltrating cell type found in both the rabbit and human corneas, were often found in intimate contact with degenerat ing keratocytes. REFERENCES 1. Pettit, T. H.: Herpes simplex keratitis: current concepts. In Becker, B., and Bürde, R. M. (eds.): Current Concepts in Ophthalmology. St. Louis, C. V. Mosby, 1969, vol. 2, p. 10.
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2. Kaufman, H. E.: Herpetic stromal disease, edi torial. Am. J. Ophthalmol. 80:1092, 1975. 3. : Systemic therapy of ocular herpes. Int. Ophthalmol. Clin. 15:163, 1975. 4. Laibson, P. R.: Corneal transplantation in the treatment of active and inactive herpes simplex keratitis. In Golden, B. (ed.): Ocular Inflammatory Disease. Springfield, Charles C Thomas, 1974, p. 297. 5. Williams, L. E., Nesburn, A. B., and Kaufman, H. E.: Experimental induction of disciform kerati tis. Arch. Ophthalmol. 73:112, 1965. 6. Swyers, J. S., Lausch, R. N., and Kaufman, H. E.: Corneal hypersensitivity to herpes simplex. Br. J. Ophthalmol. 51:843, 1967. 7. Lausch, R. N., Swyers, J. S., and Kaufman, H. E.: Delayed hypersensitivity to herpes simplex virus in the guinea pig. J. Immunol. 96:981, 1966. 8. Henson, D., Heimsen, R., Becker, K. E., Strano, A. J., Sullivan, M., and Harris, D.: Ultrastructural localization of herpes simplex virus antigens on rabbit corneal cells using sheep antihuman IgG antihorse ferritin hybrid antibodies. Invest. Oph thalmol. 13:819,1974. 9. Kaufman, H. E.: Therapy of corneal virus dis ease. In King, J. H., and McTigue, J. W. (eds.): The Cornea—World Congress. Washington, 1965, p. 187. 10. : Treatment of deep herpetic keratitis with IDU and corticosteroids. Eye Ear Nose Throat Digest. 25:37, 1963. 11. Metcalf, J. F., McNeill, J. I., and Kaufman, H. E.: Experimental disciform edema and necrotizing
DECEMBER, 1976
keratitis in the rabbit. Invest. Ophthalmol. In press. 12. Breese, S. S., Jr., and Hsu, K. D.: Techniques of ferritin-tagged antibodies. In Maramorosch, K., and Koprowski, H. (eds.): Methods in Virology, vol. 5. New York, Academic Press, 1971, p. 404. 13. Weir, D. M.: Handbook of Experimental Im munology. Oxford, Blackwell Scientific Publica tions, 1967, p. 581. 14. Goldman, M.: Fluorescent Antibody Meth ods. New York, Academic Press, 1968, p. 103. 15. Oakes, J. E.: Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection. Infect. Immun. 12:166, 1975. 16. Kanai, A., and Polack, F. M.: Ultramicroscopic changes in the corneal graft stroma during early rejection. Invest. Ophthalmol. 10:415, 1971. 17. Polack, F. M., and Kanai, A.: Electron micro scopic studies of graft endothelium in corneal graft rejection. Am. J. Ophthalmol. 73:711, 1972. 18. Gowars, J. L.: Immunology of the small lym phocyte. In Good, R. A., and Fisher, D. W. (eds.): Immunobiology. Stamford, Sinauer Associates, Inc., 1972, p. 18. 19. Kelleher, J. J., Varani, J., and Nelson, W. W.: Establishment of a nonproductive herpes simplex virus infection in rabbit kidney cells. Infect. Im mun. 12:128, 1975. 20. Green, M.: Oncogenic viruses. In Snell, E. E., Boyer, P. D., Meister, A., and Sinsheimer, R. L. (eds.): Annual Review of Biochemistry. Palo Alto, Annual Reviews, Inc., 1970, p. 701.
