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Conjunctival Deposition of Eosinophil Granule Major Basic Protein in Vernal Keratoconjunctivitis and Contact Lens-Associated Giant Papillary Conjunctivitis
Conjunctival Deposition of Eosinophil Granule Major Basic Protein in Vernal Keratoconjunctivitis and Contact Lens-Associated Giant Papillary Conjunctivitis Stefan D. Trocme, M.D., Gail M. Kephart, B.S., Mathea R. Allansmith, M.D., William M. Bourne, M.D., and Gerald J. Gleich, M.D.
To investigate the role of the eosinophil in vernal keratoconjunctivitis and contact lensassociated giant papillary conjunctivitis, we assessed the presence of eosinophil granule major basic protein in conjunctival tissues by immunofluorescence. Biopsy specimens of conjunctiva were taken from nine patients with vernal keratoconjunCtivitis, seven patients with giant papillary conjunctivitis, and five control subjects. We performed a masked semiquantitative assessment of immunofluorescence on sections from each specimen. The vernal keratoconjunctivitis and giant papillary conjunctivitis groups had significantly (P < .05) more major basic protein deposition than controls. No significant correlation between severity of disease and degree of major basic protein deposition was found. We found extracellular eosinophil granules in one of three vernal keratoconjunctivitis specimens examined by transmission electron microscopy. Thus, eosinophil degranulation commonly occurs in vernal keratoconjunctivitis and giant papillary conjunctivitis with release of eosinophil granule major basic protein and presum-
Accepted for publication March 21, 1989. From the Departments of Ophthalmology (Drs. Trocme and Bourne) and Immunology (Ms. Kephart and Dr. Gleich), Mayo Clinic, Rochester, Minnesota and the Department of Ophthalmology, Harvard Medical School and Ocular Immunology Unit, Eye Research Institute of Retina Foundation (Dr. Allansmith), Boston, Massachusetts. Supported in part by National Institutes of Health grants EY 02037, AI 09728, and AI 15231, Research to Prevent Blindness, Inc., and the Mayo Clinic and Foundation. Reprint requests to William M. Bourne, M.D., Department of Ophthalmology, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905.
©AMERICAN JOURNAL OF OPHTHALMOLOGY
108:57--63,
JULY,
ably other toxic granule proteins onto affected tissues. These cationic proteins are potent cytotoxins and are able to stimulate mast cell degranulation. VERNAL KERATOCONJUNCTIVITIS and contact lens-associated giant papillary conjunctivitis are characterized histologically by marked mast cell degranulation and eosinophil infiltration. 1,2 Patients with both conditions have giant papillae of the tarsal conjunctiva of the upper eyelid, conjunctival hyperemia, mucus formation, and itching. Components of both immediate and delayed hypersensitivity appear to be involved in the immunopathologic process of vernal keratoconjunctivitis and giant papillary conjunctivltis." Whereas vernal keratoconjunctivitis is a disease of youth and predominantly affects males," giant papillary conjunctivitis occurs only after a foreign body, such as a contact lens," suture," or ocular prosthesis," has been introduced to the ocular surface. The role of the eosinophil in hypersensitivity diseases such as vernal keratoconjunctivitis and giant papillary conjunctivitis remains obscure. It was previously thought that the eosinophil might dampen allergic inflammation by neutralizing the mediators of anaphylaxis.t-" More recent information suggests that the eosinophil is an effector of tissue damage in hypersensitivity diseases." For example, the eosinophil granule major basic protein causes desquamation of respiratory epithelium in vitro and is deposited at sites of damage in vivo. 12,13 Eosinophils may enhance immunity to helminths; major basic protein is toxic to helmlnths.P-" Major basic protein also elicits mast cell degranulation," and high levels of major basic protein and Charcot-Leyden crystal protein have been measured in tears from patients
1989
57
58
AMERICAN JOURNAL OF OPHTHALMOLOGY
with vernal keratoconjunctivitis; major basic protein levels correlated with severity of disease." The human eosinophil granule major basic protein molecule is strongly cationic and has a molecular weight of approximately 14,000. 18, 19 In the guinea pig, major basic protein accounts for more than 50% of the eosinophil granule protein and for about 25% of the total cellular protein.P-" We undertook this study (1) to determine whether eosinophil granule major basic protein is present in conjunctiva of normal subjects and patients with vernal keratoconjunctivitis and giant papillary conjunctivitis, (2) to compare the magnitude of deposition among the three groups, and (3) to determine whether the amount of conjunctival eosinophil granule major basic protein correlates with severity of disease.
Material and Methods We evaluated conjunctival tissue specimens from nine patients with vernal keratoconjunctivitis, seven patients with giant papillary conjunctivitis, and five normal control subjects. One of us (M.R.A.) assigned clinical stages (Table) and performed superior tarsal conjunctival biopsies in all patients. The stages were estimates of clinical severity according to a published staging system." Stage 1 (preclinical disease) was characterized by mild symptoms only. None of our affected patients had stage 1 disease. Stage 2 had elevation of normal papillae, initial formation of giant papillae with occasional staining with 2% fluorescein, none or mild erythema, mild sheets of mucus, and moderate itching. Stage 3 exhibited an increased number, size, and elevation of papillae; more staining of papillae; variable erythema and edema; heavy sheets of mucus; and moderate to severe itching. Stage 4 had flatten-
TABLE CLINICAL STAGES OF PATIENTS· STAGE GROUP
Vernal keratoconjunctivitis Giant papillary conjunctivitis •Stages are defined in the texl.
2
3
4
TOTAL
1 2
3 2
5 3
9 7
July, 1989
ing of the apices of the papillae, more erythema and more constant edema than stage 3, severely increased mucus and itching, and eyelids that were stuck together. All tissues were fixed and processed according to a technique described by Sainte-Marie. 22 Tissue specimens were sectioned and processed for immunofluorescence for detection of eosinophil granule major basic protein as previously described.P-" In brief, e-um sections were cut and mounted to slides with LePage's glue. Subsequent to deparaffinization in xylene and rehydration through graded ethanol solutions, the sections were digested with trypsin 0.1 % for one hour at 37 C, washed in phosphate-buffered saline (pH 7.5), and incubated overnight at 4 C in 10% normal goat serum. Slides were overlaid with either the negative control serum (normal rabbit IgG), or the test material (affinity chromatographypurified rabbit antibody to human eosinophil granule major basic protein), and incubated at 37 C for 30 minutes. The slides were then washed with phosphate-buffered saline and stained with 1% chromotope 2R, washed again with phosphate-buffered saline, overlaid with a fluorescein-conjugated IgG fraction of goat anti-rabbit IgG, and incubated for 30 minutes at 37 C. As a final step, the slides were washed with phosphate-buffered saline, mounted in a 10% phosphate-buffered saline:90% glycerol solution containing p-phenylenediamine," coverslipped, and sealed with nail polish. Slides were examined with a standard microscope equipped with vertical illumination for epifluorescence and a fluorescein filter system. Addition of p-phenylenediamine to the buffered glycerol mounting medium reduced fading, allowing for prolonged examination and multiple photographs of areas of interest. 25 A formalin-fixed nasal polyp with numerous eosinophils served as a positive control for the assay. We took three color photomicrographs of each section in order to include the total area of each specimen. Masked assessment of fluorescence in photographs of each tissue section was performed by consensus of three observers (S.D.T., G.M.K., and G.].G.). We assessed intensity of fluorescence on a scale of 0 to 3+ (0 = none; 1+ = minimal; 2+ =moderate; 3+ = maximal) and recorded the area (nearest 25%, 50%, 75%, or 100% of total area) of each section that exhibited each intensity level. We calculated the mean intensity of fluorescence for each specimen from the proportions (25%, 50%,
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ConjunctivalEosinophil Basic Protein
75%, or 100%) exhibiting each level of intensity. We used a rank sum test" to compare the fluorescent intensities of each group and a Spearman rank correlation to correlate fluorescent intensity with clinical stage. P < .05 (twotailed) was considered statistically significant. Finally, because we did not observe intact eosinophils in either the serial hematoxylin and eosin-stained or anti-eosinophil granule major basic protein-stained sections of the SainteMarie-fixed tissues, we analyzed two normal and three 3+ vernal keratoconjunctivitis specimens taken from the upper tarsal conjunctiva by transmission electron microscopy to confirm the presence of eosinophils. These tissues were obtained from patients other than those studied by immunofluorescence because suitably fixed tissue for analysis by electron microscopy was not available. The five specimens were fixed for three hours in a solution composed of 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.025% calcium chloride in 0.1 M cacodylate buffer (pH 7.4).27 After a three-hour period, the fixative was replaced with cold 0.11 M cacodylate buffer (pH 7.4) containing 10% sucrose. Tissues were postfixed in osmium tetroxide, dehydrated in ethanol, and embedded in epoxy resin. Results
The intensity of eosinophil granule major basic protein deposition in both the vernal keratoconjunctivitis and giant papillary conjunctivitis groups was significantly higher than that of the controls (Fig. 1). There was no significant difference between the vernal keratoconjunctivitis and giant papillary conjunctivitis groups, however. In tissue specimens from normal control subjects, little or no major basic protein deposition was observed, whereas in patients with vernal keratoconjunctivitis and giant papillary conjunctivitis marked major basic protein deposition was seen (Fig. 2). Most of the major basic protein was diffusely deposited in tissues and was not confined to cells. Only one specimen from a control individual had a relatively high mean intensity of major basic protein deposition (0.75); this resulted from one isolated focus of 3+ staining in a section with a small total area. No significant correlation between mean intensity of eosinophil granule major basic protein deposition and severity of disease was
59
2.0
11 ~
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1.0
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0.5
444 44
•••
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Giant papillary
4
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KBr8Ioconjunctlvllls
•
•
•
Control
conJunctlvttis
Fig. 1 (Trocme and associates). Mean intensity of eosinophil granule major basic protein fluorescence for each individual in the vernal keratoconjunctivitis, giant papillary conjunctivitis, and control groups. The giant papillary conjunctivitis and vernal keratoconjunctivitis groups each had significantly higher fluorescence intensities than the controls (P < .05). There was no significant difference in fluorescence intensity between the vernal keratoconjunctivitis and giant papillary conjunctivitis groups.
found for either the vernal keratoconjunctivitis or the giant papillary conjunctivitis group (Fig. 3), and, indeed, a trend of less major basic protein deposition in stage 4 than in stage 3 disease was noted for both groups. The difference between stage 3 and 4 disease was significant (P < .05) only if the vernal keratoconjunctivitis and giant papillary conjunctivitis groups were combined. Finally, in the electron microscopy specimens, occasional intact eosinophils and mast cells were observed in the tissue specimens from normal individuals. Numerous intact eosinophils and mast cells were seen in all three of the vernal keratoconjunctivitis specimens; extensive eosinophil degranulation was observed in one of them (Fig. 4). Discussion
Previous studies have demonstrated that assessment of eosinophil involvement in disease cannot be based simply on numbers of eosinophils in tissue but must include assessment of eosinophil degranulation, which can be measured by immunofluorescence localization of the eosinophil granule major basic protein. 28 We observed marked major basic protein deposition with few intact eosinophils. The substan-
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AMERICAN JOURNAL OF OPHTHALMOLOGY
July, 1989
•
Fig. 2 (Trocme and associates). Eosinophil granule major basic protein deposition in conjunctivae. Top left, Section of control conjunctival tissue stained with hematoxylin and eosin. Section serial to top left stained by immunofluorescence for eosinophil granule major basic protein demonstrated no fluorescence. Middle left, Section of 3+ giant papillary conjunctivitis conjunctiva stained with hematoxylin and eosin. Note the accumulation of inflammatory cells in the substantia propria. Middle right, Section serial to middle left stained by immunofluorescence for eosinophil granule major basic protein and displaying 25% 0, 25% 1+, and 50% 2+ staining. The mean intensity of fluorescence of the entire specimen, based on evaluation of one additional photograph, was 0.58. Bottom left, Section of 3+ vernal keratoconjunctivitis conjunctiva stained with hematoxylin and eosin. Note the papilla formation and subepithelial inflammatory infiltrate. Bottom right, Section serial to that in bottom left stained by immunofluorescence for eosinophil granule major basic protein and showing 25% 1 + and 75% 2+ staining. The mean intensity of the entire specimen, based on the evaluation of two additional photographs, was 0.61 (all photomicrographs, x 160).
Vol. 108, No. 1
2.0
~~ G)
1.5
II)
~ 0~ .m;;::l ~
:E
'5
Conjunctival Eosinophil Basic Protein
• Vemal Keratoconjunctivltis • Giant papillary conjunctivitis
1.0
0.5
0
•• 2
• •• •• 3
•
• ••• •
•• 4
Clinical stage Fig. 3 (Trocme and associates). Correlation between mean intensity of eosinophil granule major basic protein fluorescence and severity of disease for the vernal keratoconjunctivitis and giant papillary conjunctivitis groups. No significant correlation was observed. A trend of less fluorescence in stage 4 than in stage 3 disease is evident, however. This difference in fluorescence between stage 3 and stage 4 is significant (P < .05) when data from both groups are combined.
tial deposition of major basic protein suggests that conjunctival eosinophil degranulation occurs in both vernal keratoconjunctivitis and giant papillary conjunctivitis. We presume also
61
that other toxic cationic eosinophil granule proteins, including the eosinophil peroxidase, the eosinophil-derived neurotoxin, and the eosinophil cationic protein, are deposited into the conjunctivae of patients with vernal keratoconjunctivitis and giant papillary conjunctivitis. II Eosinophil granule major basic protein deposition in conjunctival tissues may playa role in the pathogenesis of vernal keratoconjunctivitis and giant papillary conjunctivitis. In vitro experiments have demonstrated that major basic protein induces noncytotoxic histamine secretion from rat mast cells and human basophils": the release of major basic protein into the conjunctiva may in part explain the extensive mast cell degranulation described in both vernal keratoconjunctivitis and giant papillary conjunctivitis." and the cytotoxic effects of major basic protein may enhance the conjunctival inflammatory reaction. Organ culture studies on guinea pig trachea have shown that major basic protein causes desquamation and damage of respiratory epithelial cells":": incubation with major basic protein impairs ciliary activity and causes the respiratory epithelium to slough." The observed trend of less major basic protein deposition in stage 4 than in stage 3 disease cannot be easily explained. We speculate that the eosinophil activity may be higher
Fig. 4 (Trocme and associates). Electron photomicrographs of free eosinophil granules from a patient with 3+ vernal keratoconjunctivitis that show numerous crystalloidcontaining granules which are not confined to an intact cell. Inset, Enlargement of a portion of the area shown in box. The solid arrowhead identifies an eosinophil granule with the typical dense core and less dense matrix. The granule identified by the open arrowhead appears to be dissolving (x 4,500; inset, x 10,000).
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AMERICAN JOURNAL OF OPHTHALMOLOGY
in disease of intermediate severity and may decrease after tissue injury has occurred and the proliferative reaction (formation of papillae) progresses. Although the present study indicates that eosinophil granule major basic protein is present in conjunctival tissues of patients with either vernal keratoconjunctivitis or giant papillary conjunctivitis, a previous study demonstrated major basic protein in tears from patients with vernal keratoconjunctivitis but not in tears from patients with giant papillary conjunctivitis." Thus, if major basic protein is present in the tears of patients with giant papillary conjunctivitis, it must be present in small amounts. Because high levels of major basic protein have been detected in tears from patients with vernal keratoconjunctivitis, it has been suggested that major basic protein may participate in the formation of vernal shield ulcers." Tear major basic protein and contact lens interactions could be of clinical importance. A recent study indicates that basic tear proteins adhere to contact lenses." If eosinophil granule major basic protein is present in tears, deposits with potentially toxic effects could accumulate over time.
References 1. Morgan, G.: The pathology of vernal conjunctivitis. Trans. Ophthalmol. Soc. U.K. 91:467, 1971. 2. Allansmith, M. R., Baird, R. 5., and Greiner, J. V.: Vernal conjunctivitis and contact lensassociated giant papillary conjunctivitis compared and contrasted. Am. J. Ophthalmol. 87:544, 1979. 3. Allansmith, M. R.: The Eye and Immunology. St. Louis, C. V. Mosby, 1982, pp. 118-130. 4. Allansmith, M. R., and Frick, O. L.: Antibodies to grass in vernal conjunctivitis. J. Allergy 34:535, 1963. 5. Allansmith, M. R., Korb, D. R., Greiner, J. V., Henriquez, A.S., Simon, M. A., and Finnemore, V. M.: Giant papillary conjunctivitis in contact lens wearers. Am. J. Ophthalmol. 83:697, 1977. 6. Sugar, A., and Meyer, R. F.: Giant papillary conjunctivitis after keratoplasty. Am. J. Ophthalmol. 91:239, 1981. 7. Meisler, D. M., Krachmer, J. H., and Goeken, J. A.: An immunopathologic study of giant papillary conjunctivitis associated with an ocular prosthesis. Am. J. Ophthalmol. 92:368, 1981. 8. Bass, D. A.: The function of eosinophils. Ann. Intern. Med. 91:120, 1979. 9. Goetzl, E. J., Wasserman, S. I., and Austen, K. E.: Eosinophil polymorphonuclear leucocyte
July, 1989
function in immediate hypersensitivity. Arch. PathoI. 99:1, 1975. 10. Hubscher, T.: Role of the eosinophils in the allergic reaction. I. ED!. An eosinophil-derived inhibitor of histamine release. J. Immunol. 114:1379, 1975. 11. Gleich, G. J., and Adolphson, C. R: The eosinophilic leukocyte. Structure and function. Adv. Immunol. 39:177, 1986. 12. Frigas, E., and Gleich, G. J.: The eosinophil and the pathophysiology of asthma. J. Allergy Clin. Immunol. 77:527, 1986. 13. Harlin, S. L., Ansel, D. G., Lane, S. R., Myers, J., Kephart, G. M., and Gleich, G. J.: A clinical and pathological study of chronic sinusitis. The role of the eosinophil. J. Allergy Clin. Immunol. 81:867, 1988. 14. Butterworth, A. E., Wassom, D. L., Gleich, G. J., Loegering, D. A., and David, J. R.: Damage to schistosomula of Schistosoma mansoni induced directly by eosinophil major basic protein. J. Immunol. 122:221, 1979. 15. Wassom, D. L., and Gleich, G. J.: Damage to Trichinella spiralis newborn larvae by eosinophil major basic protein. Am. J. Trop. Med. 28:860, 1979. 16. O'Donnell, M. c.. Ackerman, S. J., Gleich, G. J., and Thomas, L. L.: Alteration of basophil and mast cell histamine release by eosinophil granule major basic protein. J. Exp. Med. 157:1981, 1985. 17. Udell, I. J., Gleich, G. J., Allansmith, M. R., Ackerman, S. J., and Abelson, M. B.: Eosinophil granule major basic protein and Charcot-Leyden crystal protein in human tears. Am. J. Ophthalmol. 92:824, 1981. 18. Wasmoen, T. L., Bell, M. P., Loegering, D. A., Prendergast, F. G., and McKean, D. J.: Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein. J. BioI. Chem. 263:12559, 1988. 19. Barker, R. L., Gleich, G. J., and Pease, L. R.: Acidic precursor revealed in human eosinophil granule major basic protein eDNA. J. Exp. Med. 168:1493, 1988. 20. Gleich, G. J., Loegering, D. A., and Maldonado, J. E.: Identification of a major basic protein in guinea pig eosinophil granules. J. Exp. Med. 137:1459, 1973. 21. Archer, G. T., and Hirsch, J. G.: Isolation of granules from eosinophil leucocytes and study of their enzyme content. J. Exp. Med. 140:313, 1974. 22. Sainte-Marie, G.: A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10:250, 1962. 23. Filley, W. V., Holley, K. E., Kephart, G. M., and Gleich, G. J.: Identification by immunofluorescence of eosinophil granule major basic protein in lung tissues of patients with bronchial asthma. Lancet 2:11, 1982. 24. Peters, M. S., Schroeter, A. L., Kephart, G. M., and Gleich, G. J.: Immunofluorescence identification of eosinophil granule major basic protein in chronic urticaria. J. Invest. Dermatol. 81:39, 1983.
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25. Johnson, G. D., and de C. Nogueira Araujo, G. M.: A simple method of reducing the fading of immunofluorescence during microscopy. J. Immuno!. Methods 43:349, 1981. 26. Moses, L. E., Emerson, ]. D., and Hosseini, N.: Analyzing data from ordered categories. N. Eng!. J. Med. 311:442, 1984. 27. Karnovsky, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Bio!. 27:1372, 1965.
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28. Leiferman, K. M., Ackerman, S. J., Sampson, H. A., Haugen, H. S., Venenci, P. Y., and Gleich, G. J.: Dermal deposition of eosinophil-granule major basic protein in atopic dermatitis. Comparison with onchocerciasis. N. Eng!. J. Med. 313:282, 1985. 29. Sach, R. A., Jones, B., Antignani, A., Libow, R., and Harvey, H.: Specificity and biological activity of the protein deposited on the hydrogel surface. Relationship of polymer structure to biofilm formation. Invest. Ophthalmol. Vis. Sci. 28:842, 1987.
OPHTHALMIC MINIATURE
The man who preserved his sight the longest, recovered the soonest. To his exertions alone we owe that we are now within a few leagues of Guadeloupe, this 21st day of June, 1819. I am almost well. The surgeon and eleven more are irrecoverably blind. The captain has lost one eye. Four others have met with the same calamity. Five are able to see, though dimly, with both. Among the slaves, 39 are completely blind and the rest blind of one eye or their sight otherwise injured ... Isidor Paiewonsky, Eyewitness Accounts of Slavery in the Danish West Indies Privately printed by the author, 1987, p. 88
To investigate the role of the eosinophil in vernal keratoconjunctivitis and contact lensassociated giant papillary conjunctivitis, we assessed the presence of eosinophil granule major basic protein in conjunctival tissues by immunofluorescence. Biopsy specimens of conjunctiva were taken from nine patients with vernal keratoconjunCtivitis, seven patients with giant papillary conjunctivitis, and five control subjects. We performed a masked semiquantitative assessment of immunofluorescence on sections from each specimen. The vernal keratoconjunctivitis and giant papillary conjunctivitis groups had significantly (P < .05) more major basic protein deposition than controls. No significant correlation between severity of disease and degree of major basic protein deposition was found. We found extracellular eosinophil granules in one of three vernal keratoconjunctivitis specimens examined by transmission electron microscopy. Thus, eosinophil degranulation commonly occurs in vernal keratoconjunctivitis and giant papillary conjunctivitis with release of eosinophil granule major basic protein and presum-
Accepted for publication March 21, 1989. From the Departments of Ophthalmology (Drs. Trocme and Bourne) and Immunology (Ms. Kephart and Dr. Gleich), Mayo Clinic, Rochester, Minnesota and the Department of Ophthalmology, Harvard Medical School and Ocular Immunology Unit, Eye Research Institute of Retina Foundation (Dr. Allansmith), Boston, Massachusetts. Supported in part by National Institutes of Health grants EY 02037, AI 09728, and AI 15231, Research to Prevent Blindness, Inc., and the Mayo Clinic and Foundation. Reprint requests to William M. Bourne, M.D., Department of Ophthalmology, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905.
©AMERICAN JOURNAL OF OPHTHALMOLOGY
108:57--63,
JULY,
ably other toxic granule proteins onto affected tissues. These cationic proteins are potent cytotoxins and are able to stimulate mast cell degranulation. VERNAL KERATOCONJUNCTIVITIS and contact lens-associated giant papillary conjunctivitis are characterized histologically by marked mast cell degranulation and eosinophil infiltration. 1,2 Patients with both conditions have giant papillae of the tarsal conjunctiva of the upper eyelid, conjunctival hyperemia, mucus formation, and itching. Components of both immediate and delayed hypersensitivity appear to be involved in the immunopathologic process of vernal keratoconjunctivitis and giant papillary conjunctivltis." Whereas vernal keratoconjunctivitis is a disease of youth and predominantly affects males," giant papillary conjunctivitis occurs only after a foreign body, such as a contact lens," suture," or ocular prosthesis," has been introduced to the ocular surface. The role of the eosinophil in hypersensitivity diseases such as vernal keratoconjunctivitis and giant papillary conjunctivitis remains obscure. It was previously thought that the eosinophil might dampen allergic inflammation by neutralizing the mediators of anaphylaxis.t-" More recent information suggests that the eosinophil is an effector of tissue damage in hypersensitivity diseases." For example, the eosinophil granule major basic protein causes desquamation of respiratory epithelium in vitro and is deposited at sites of damage in vivo. 12,13 Eosinophils may enhance immunity to helminths; major basic protein is toxic to helmlnths.P-" Major basic protein also elicits mast cell degranulation," and high levels of major basic protein and Charcot-Leyden crystal protein have been measured in tears from patients
1989
57
58
AMERICAN JOURNAL OF OPHTHALMOLOGY
with vernal keratoconjunctivitis; major basic protein levels correlated with severity of disease." The human eosinophil granule major basic protein molecule is strongly cationic and has a molecular weight of approximately 14,000. 18, 19 In the guinea pig, major basic protein accounts for more than 50% of the eosinophil granule protein and for about 25% of the total cellular protein.P-" We undertook this study (1) to determine whether eosinophil granule major basic protein is present in conjunctiva of normal subjects and patients with vernal keratoconjunctivitis and giant papillary conjunctivitis, (2) to compare the magnitude of deposition among the three groups, and (3) to determine whether the amount of conjunctival eosinophil granule major basic protein correlates with severity of disease.
Material and Methods We evaluated conjunctival tissue specimens from nine patients with vernal keratoconjunctivitis, seven patients with giant papillary conjunctivitis, and five normal control subjects. One of us (M.R.A.) assigned clinical stages (Table) and performed superior tarsal conjunctival biopsies in all patients. The stages were estimates of clinical severity according to a published staging system." Stage 1 (preclinical disease) was characterized by mild symptoms only. None of our affected patients had stage 1 disease. Stage 2 had elevation of normal papillae, initial formation of giant papillae with occasional staining with 2% fluorescein, none or mild erythema, mild sheets of mucus, and moderate itching. Stage 3 exhibited an increased number, size, and elevation of papillae; more staining of papillae; variable erythema and edema; heavy sheets of mucus; and moderate to severe itching. Stage 4 had flatten-
TABLE CLINICAL STAGES OF PATIENTS· STAGE GROUP
Vernal keratoconjunctivitis Giant papillary conjunctivitis •Stages are defined in the texl.
2
3
4
TOTAL
1 2
3 2
5 3
9 7
July, 1989
ing of the apices of the papillae, more erythema and more constant edema than stage 3, severely increased mucus and itching, and eyelids that were stuck together. All tissues were fixed and processed according to a technique described by Sainte-Marie. 22 Tissue specimens were sectioned and processed for immunofluorescence for detection of eosinophil granule major basic protein as previously described.P-" In brief, e-um sections were cut and mounted to slides with LePage's glue. Subsequent to deparaffinization in xylene and rehydration through graded ethanol solutions, the sections were digested with trypsin 0.1 % for one hour at 37 C, washed in phosphate-buffered saline (pH 7.5), and incubated overnight at 4 C in 10% normal goat serum. Slides were overlaid with either the negative control serum (normal rabbit IgG), or the test material (affinity chromatographypurified rabbit antibody to human eosinophil granule major basic protein), and incubated at 37 C for 30 minutes. The slides were then washed with phosphate-buffered saline and stained with 1% chromotope 2R, washed again with phosphate-buffered saline, overlaid with a fluorescein-conjugated IgG fraction of goat anti-rabbit IgG, and incubated for 30 minutes at 37 C. As a final step, the slides were washed with phosphate-buffered saline, mounted in a 10% phosphate-buffered saline:90% glycerol solution containing p-phenylenediamine," coverslipped, and sealed with nail polish. Slides were examined with a standard microscope equipped with vertical illumination for epifluorescence and a fluorescein filter system. Addition of p-phenylenediamine to the buffered glycerol mounting medium reduced fading, allowing for prolonged examination and multiple photographs of areas of interest. 25 A formalin-fixed nasal polyp with numerous eosinophils served as a positive control for the assay. We took three color photomicrographs of each section in order to include the total area of each specimen. Masked assessment of fluorescence in photographs of each tissue section was performed by consensus of three observers (S.D.T., G.M.K., and G.].G.). We assessed intensity of fluorescence on a scale of 0 to 3+ (0 = none; 1+ = minimal; 2+ =moderate; 3+ = maximal) and recorded the area (nearest 25%, 50%, 75%, or 100% of total area) of each section that exhibited each intensity level. We calculated the mean intensity of fluorescence for each specimen from the proportions (25%, 50%,
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ConjunctivalEosinophil Basic Protein
75%, or 100%) exhibiting each level of intensity. We used a rank sum test" to compare the fluorescent intensities of each group and a Spearman rank correlation to correlate fluorescent intensity with clinical stage. P < .05 (twotailed) was considered statistically significant. Finally, because we did not observe intact eosinophils in either the serial hematoxylin and eosin-stained or anti-eosinophil granule major basic protein-stained sections of the SainteMarie-fixed tissues, we analyzed two normal and three 3+ vernal keratoconjunctivitis specimens taken from the upper tarsal conjunctiva by transmission electron microscopy to confirm the presence of eosinophils. These tissues were obtained from patients other than those studied by immunofluorescence because suitably fixed tissue for analysis by electron microscopy was not available. The five specimens were fixed for three hours in a solution composed of 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.025% calcium chloride in 0.1 M cacodylate buffer (pH 7.4).27 After a three-hour period, the fixative was replaced with cold 0.11 M cacodylate buffer (pH 7.4) containing 10% sucrose. Tissues were postfixed in osmium tetroxide, dehydrated in ethanol, and embedded in epoxy resin. Results
The intensity of eosinophil granule major basic protein deposition in both the vernal keratoconjunctivitis and giant papillary conjunctivitis groups was significantly higher than that of the controls (Fig. 1). There was no significant difference between the vernal keratoconjunctivitis and giant papillary conjunctivitis groups, however. In tissue specimens from normal control subjects, little or no major basic protein deposition was observed, whereas in patients with vernal keratoconjunctivitis and giant papillary conjunctivitis marked major basic protein deposition was seen (Fig. 2). Most of the major basic protein was diffusely deposited in tissues and was not confined to cells. Only one specimen from a control individual had a relatively high mean intensity of major basic protein deposition (0.75); this resulted from one isolated focus of 3+ staining in a section with a small total area. No significant correlation between mean intensity of eosinophil granule major basic protein deposition and severity of disease was
59
2.0
11 ~
ii
~o
•
1.5
1.0
444
0.5
444 44
•••
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Giant papillary
4
0
KBr8Ioconjunctlvllls
•
•
•
Control
conJunctlvttis
Fig. 1 (Trocme and associates). Mean intensity of eosinophil granule major basic protein fluorescence for each individual in the vernal keratoconjunctivitis, giant papillary conjunctivitis, and control groups. The giant papillary conjunctivitis and vernal keratoconjunctivitis groups each had significantly higher fluorescence intensities than the controls (P < .05). There was no significant difference in fluorescence intensity between the vernal keratoconjunctivitis and giant papillary conjunctivitis groups.
found for either the vernal keratoconjunctivitis or the giant papillary conjunctivitis group (Fig. 3), and, indeed, a trend of less major basic protein deposition in stage 4 than in stage 3 disease was noted for both groups. The difference between stage 3 and 4 disease was significant (P < .05) only if the vernal keratoconjunctivitis and giant papillary conjunctivitis groups were combined. Finally, in the electron microscopy specimens, occasional intact eosinophils and mast cells were observed in the tissue specimens from normal individuals. Numerous intact eosinophils and mast cells were seen in all three of the vernal keratoconjunctivitis specimens; extensive eosinophil degranulation was observed in one of them (Fig. 4). Discussion
Previous studies have demonstrated that assessment of eosinophil involvement in disease cannot be based simply on numbers of eosinophils in tissue but must include assessment of eosinophil degranulation, which can be measured by immunofluorescence localization of the eosinophil granule major basic protein. 28 We observed marked major basic protein deposition with few intact eosinophils. The substan-
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•
Fig. 2 (Trocme and associates). Eosinophil granule major basic protein deposition in conjunctivae. Top left, Section of control conjunctival tissue stained with hematoxylin and eosin. Section serial to top left stained by immunofluorescence for eosinophil granule major basic protein demonstrated no fluorescence. Middle left, Section of 3+ giant papillary conjunctivitis conjunctiva stained with hematoxylin and eosin. Note the accumulation of inflammatory cells in the substantia propria. Middle right, Section serial to middle left stained by immunofluorescence for eosinophil granule major basic protein and displaying 25% 0, 25% 1+, and 50% 2+ staining. The mean intensity of fluorescence of the entire specimen, based on evaluation of one additional photograph, was 0.58. Bottom left, Section of 3+ vernal keratoconjunctivitis conjunctiva stained with hematoxylin and eosin. Note the papilla formation and subepithelial inflammatory infiltrate. Bottom right, Section serial to that in bottom left stained by immunofluorescence for eosinophil granule major basic protein and showing 25% 1 + and 75% 2+ staining. The mean intensity of the entire specimen, based on the evaluation of two additional photographs, was 0.61 (all photomicrographs, x 160).
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2.0
~~ G)
1.5
II)
~ 0~ .m;;::l ~
:E
'5
Conjunctival Eosinophil Basic Protein
• Vemal Keratoconjunctivltis • Giant papillary conjunctivitis
1.0
0.5
0
•• 2
• •• •• 3
•
• ••• •
•• 4
Clinical stage Fig. 3 (Trocme and associates). Correlation between mean intensity of eosinophil granule major basic protein fluorescence and severity of disease for the vernal keratoconjunctivitis and giant papillary conjunctivitis groups. No significant correlation was observed. A trend of less fluorescence in stage 4 than in stage 3 disease is evident, however. This difference in fluorescence between stage 3 and stage 4 is significant (P < .05) when data from both groups are combined.
tial deposition of major basic protein suggests that conjunctival eosinophil degranulation occurs in both vernal keratoconjunctivitis and giant papillary conjunctivitis. We presume also
61
that other toxic cationic eosinophil granule proteins, including the eosinophil peroxidase, the eosinophil-derived neurotoxin, and the eosinophil cationic protein, are deposited into the conjunctivae of patients with vernal keratoconjunctivitis and giant papillary conjunctivitis. II Eosinophil granule major basic protein deposition in conjunctival tissues may playa role in the pathogenesis of vernal keratoconjunctivitis and giant papillary conjunctivitis. In vitro experiments have demonstrated that major basic protein induces noncytotoxic histamine secretion from rat mast cells and human basophils": the release of major basic protein into the conjunctiva may in part explain the extensive mast cell degranulation described in both vernal keratoconjunctivitis and giant papillary conjunctivitis." and the cytotoxic effects of major basic protein may enhance the conjunctival inflammatory reaction. Organ culture studies on guinea pig trachea have shown that major basic protein causes desquamation and damage of respiratory epithelial cells":": incubation with major basic protein impairs ciliary activity and causes the respiratory epithelium to slough." The observed trend of less major basic protein deposition in stage 4 than in stage 3 disease cannot be easily explained. We speculate that the eosinophil activity may be higher
Fig. 4 (Trocme and associates). Electron photomicrographs of free eosinophil granules from a patient with 3+ vernal keratoconjunctivitis that show numerous crystalloidcontaining granules which are not confined to an intact cell. Inset, Enlargement of a portion of the area shown in box. The solid arrowhead identifies an eosinophil granule with the typical dense core and less dense matrix. The granule identified by the open arrowhead appears to be dissolving (x 4,500; inset, x 10,000).
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in disease of intermediate severity and may decrease after tissue injury has occurred and the proliferative reaction (formation of papillae) progresses. Although the present study indicates that eosinophil granule major basic protein is present in conjunctival tissues of patients with either vernal keratoconjunctivitis or giant papillary conjunctivitis, a previous study demonstrated major basic protein in tears from patients with vernal keratoconjunctivitis but not in tears from patients with giant papillary conjunctivitis." Thus, if major basic protein is present in the tears of patients with giant papillary conjunctivitis, it must be present in small amounts. Because high levels of major basic protein have been detected in tears from patients with vernal keratoconjunctivitis, it has been suggested that major basic protein may participate in the formation of vernal shield ulcers." Tear major basic protein and contact lens interactions could be of clinical importance. A recent study indicates that basic tear proteins adhere to contact lenses." If eosinophil granule major basic protein is present in tears, deposits with potentially toxic effects could accumulate over time.
References 1. Morgan, G.: The pathology of vernal conjunctivitis. Trans. Ophthalmol. Soc. U.K. 91:467, 1971. 2. Allansmith, M. R., Baird, R. 5., and Greiner, J. V.: Vernal conjunctivitis and contact lensassociated giant papillary conjunctivitis compared and contrasted. Am. J. Ophthalmol. 87:544, 1979. 3. Allansmith, M. R.: The Eye and Immunology. St. Louis, C. V. Mosby, 1982, pp. 118-130. 4. Allansmith, M. R., and Frick, O. L.: Antibodies to grass in vernal conjunctivitis. J. Allergy 34:535, 1963. 5. Allansmith, M. R., Korb, D. R., Greiner, J. V., Henriquez, A.S., Simon, M. A., and Finnemore, V. M.: Giant papillary conjunctivitis in contact lens wearers. Am. J. Ophthalmol. 83:697, 1977. 6. Sugar, A., and Meyer, R. F.: Giant papillary conjunctivitis after keratoplasty. Am. J. Ophthalmol. 91:239, 1981. 7. Meisler, D. M., Krachmer, J. H., and Goeken, J. A.: An immunopathologic study of giant papillary conjunctivitis associated with an ocular prosthesis. Am. J. Ophthalmol. 92:368, 1981. 8. Bass, D. A.: The function of eosinophils. Ann. Intern. Med. 91:120, 1979. 9. Goetzl, E. J., Wasserman, S. I., and Austen, K. E.: Eosinophil polymorphonuclear leucocyte
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function in immediate hypersensitivity. Arch. PathoI. 99:1, 1975. 10. Hubscher, T.: Role of the eosinophils in the allergic reaction. I. ED!. An eosinophil-derived inhibitor of histamine release. J. Immunol. 114:1379, 1975. 11. Gleich, G. J., and Adolphson, C. R: The eosinophilic leukocyte. Structure and function. Adv. Immunol. 39:177, 1986. 12. Frigas, E., and Gleich, G. J.: The eosinophil and the pathophysiology of asthma. J. Allergy Clin. Immunol. 77:527, 1986. 13. Harlin, S. L., Ansel, D. G., Lane, S. R., Myers, J., Kephart, G. M., and Gleich, G. J.: A clinical and pathological study of chronic sinusitis. The role of the eosinophil. J. Allergy Clin. Immunol. 81:867, 1988. 14. Butterworth, A. E., Wassom, D. L., Gleich, G. J., Loegering, D. A., and David, J. R.: Damage to schistosomula of Schistosoma mansoni induced directly by eosinophil major basic protein. J. Immunol. 122:221, 1979. 15. Wassom, D. L., and Gleich, G. J.: Damage to Trichinella spiralis newborn larvae by eosinophil major basic protein. Am. J. Trop. Med. 28:860, 1979. 16. O'Donnell, M. c.. Ackerman, S. J., Gleich, G. J., and Thomas, L. L.: Alteration of basophil and mast cell histamine release by eosinophil granule major basic protein. J. Exp. Med. 157:1981, 1985. 17. Udell, I. J., Gleich, G. J., Allansmith, M. R., Ackerman, S. J., and Abelson, M. B.: Eosinophil granule major basic protein and Charcot-Leyden crystal protein in human tears. Am. J. Ophthalmol. 92:824, 1981. 18. Wasmoen, T. L., Bell, M. P., Loegering, D. A., Prendergast, F. G., and McKean, D. J.: Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein. J. BioI. Chem. 263:12559, 1988. 19. Barker, R. L., Gleich, G. J., and Pease, L. R.: Acidic precursor revealed in human eosinophil granule major basic protein eDNA. J. Exp. Med. 168:1493, 1988. 20. Gleich, G. J., Loegering, D. A., and Maldonado, J. E.: Identification of a major basic protein in guinea pig eosinophil granules. J. Exp. Med. 137:1459, 1973. 21. Archer, G. T., and Hirsch, J. G.: Isolation of granules from eosinophil leucocytes and study of their enzyme content. J. Exp. Med. 140:313, 1974. 22. Sainte-Marie, G.: A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10:250, 1962. 23. Filley, W. V., Holley, K. E., Kephart, G. M., and Gleich, G. J.: Identification by immunofluorescence of eosinophil granule major basic protein in lung tissues of patients with bronchial asthma. Lancet 2:11, 1982. 24. Peters, M. S., Schroeter, A. L., Kephart, G. M., and Gleich, G. J.: Immunofluorescence identification of eosinophil granule major basic protein in chronic urticaria. J. Invest. Dermatol. 81:39, 1983.
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25. Johnson, G. D., and de C. Nogueira Araujo, G. M.: A simple method of reducing the fading of immunofluorescence during microscopy. J. Immuno!. Methods 43:349, 1981. 26. Moses, L. E., Emerson, ]. D., and Hosseini, N.: Analyzing data from ordered categories. N. Eng!. J. Med. 311:442, 1984. 27. Karnovsky, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Bio!. 27:1372, 1965.
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28. Leiferman, K. M., Ackerman, S. J., Sampson, H. A., Haugen, H. S., Venenci, P. Y., and Gleich, G. J.: Dermal deposition of eosinophil-granule major basic protein in atopic dermatitis. Comparison with onchocerciasis. N. Eng!. J. Med. 313:282, 1985. 29. Sach, R. A., Jones, B., Antignani, A., Libow, R., and Harvey, H.: Specificity and biological activity of the protein deposited on the hydrogel surface. Relationship of polymer structure to biofilm formation. Invest. Ophthalmol. Vis. Sci. 28:842, 1987.
OPHTHALMIC MINIATURE
The man who preserved his sight the longest, recovered the soonest. To his exertions alone we owe that we are now within a few leagues of Guadeloupe, this 21st day of June, 1819. I am almost well. The surgeon and eleven more are irrecoverably blind. The captain has lost one eye. Four others have met with the same calamity. Five are able to see, though dimly, with both. Among the slaves, 39 are completely blind and the rest blind of one eye or their sight otherwise injured ... Isidor Paiewonsky, Eyewitness Accounts of Slavery in the Danish West Indies Privately printed by the author, 1987, p. 88