- Email: [email protected]
IgG Specific Antibodies to Rye Grass and Ragweed Pollen Antigens in the Tear Secretions of Patients with Vernal Conjunctivitis
I G G SPECIFIC ANTIBODIES TO RYE GRASS AND RAGWEED POLLEN ANTIGENS IN THE TEAR SECRETIONS OF PATIENTS WITH VERNAL CONJUNCTIVITIS MARK BALLOW, M.D., PETER C. DONSHIK, M.D., Louis MENDELSON, PAMELA RAPACZ, B.S., AND KENNETH SPARKS, B.S. Farmington, Connecticut
M.D.,
We studied the tears of 30 patients (25 males and five females, ranging in age from 8 to 34 years) with vernal conjunctivitis for pollen-specific IgG antibodies to rye grass and ragweed antigen E by an enzyme-linked immunosorbent assay (ELISA). Eighteen of 30 (60%) and 20 of 30 (67%) patients with vernal conjunctivitis had significant levels (more than 2 S.D. from the mean of control tears) of IgG antibodies in their tear secretions to rye grass (geometric mean = 68.7 ELISA units) and ragweed antigen E (geometric mean = 50 ELISA units), respective ly. In contrast, the control groups (eight atopic individuals, four with seasonal rhinitis, and 12 with allergic conjunctivitis) had low amounts of specific IgG antibodies to these two pollen antigens in their tears. Total IgG and IgM were also increased in the tears of patients with vernal conjunctivitis. To evaluate whether these immunoglobulins and specific IgG antibodies were locally produced by the conjunctival tissues, we used transferrin as a marker for the leakage of plasma proteins into tears. We found that the specific IgG antibodies to rye grass or ragweed antigen E, or both, in the tears were locally produced by the conjuncti val tissues. The local production ranged from 20% to 99.9%. Of 17 patients with vernal conjunctivitis and undetectable pollen-specific IgE antibodies in their tears, 14 (82.4%) had tear specific IgG antibodies to rye grass or ragweed antigen E or both, whereas of those with measurable tear IgE antibodies, only nine of 13 (69%) had tear IgG antibodies to rye grass or antigen E or both. These results suggested that both IgE- and IgG-mediated immune mechanisms may be impor tant in the pathogenesis of vernal conjunctivitis. Vernal conjunctivitis, a bilateral, often severe inflammatory condition of the con junctiva, is characterized by itching, tearAccepted for publication Oct. 25, 1982. From the Departments of Pediatrics (Drs. Ballow and Mendelson, Mrs. Rapacz, and Mr. Sparks) and Ophthalmology (Dr. Donshik), University of Connec ticut Health Center School of Medicine, Farmington, Connecticut. This study was supported by grant EY-02036 from the National Eye Institute (Dr. Ballow). Reprint requests to Mark Ballow, M.D., Depart ment of Pediatrics, University of Connecticut Health Center, Farmington, CT 06032.
ing, and photophobia. Papillary hypertro phy of the upper tarsal conjunctiva and the presence of eosinophils in tear secre tions are pathognomonic of this condi tion. These clinical manifestations and the studies by Allansmith and associates1"3 and others4*6 suggest that vernal conjunc tivitis is an atopic ocular disorder. Fur ther support for an IgE-mediated patho genesis was recently reported in studies from our laboratory in which specific IgE antibodies to pollen allergens were dem onstrated in the tear secretions of more
©AMERICAN JOURNAL OF OPHTHALMOLOGY 95:161-168, 1983
161
162
AMERICAN JOURNAL OF OPHTHALMOLOGY
than one half the patients with vernal conjunctivitis studied. 7 However, the complex histopathology 3,6,8 suggests that other immune mechanisms may play a role in the pathogenesis of vernal con junctivitis. We measured specific IgG antibodies in the tears to two inhalant pollen aller gens, rye grass and ragweed antigen E, in patients with vernal conjunctivitis and in control subjects. We also investigated whether these specific IgG antibodies were locally produced by the conjunctival tissues of the eye. Our finding of locally produced pollen-specific IgG antibody together with our previous studies 7 sug gested that both IgE- and IgG-mediated immune mechanisms are involved in the pathogenesis of vernal conjunctivitis. S U B J E C T S AND M E T H O D S
Thirty patients (25 males and five fe males) with vernal conjunctivitis had the typical symptoms and physical findings of vernal conjunctivitis and had had their disease two or more seasons. The patients ranged in age from 8 to 34 years (mean, 13 years). Thirteen patients also had other allergic disorders, mostly hay fever and eczema. Only seven of 24 patients who underwent skin testing for immedi ate hypersensitivity had positive skin re activity to inhalant pollen allergens. Control groups included eight nonatopic individuals, four individuals with seasonal rhinitis, and 12 patients with allergic conjunctivitis. Tear samples were obtained approximately three to four weeks after peak symptomatology and at least two weeks after topical corticosteroids had been discontinued. Methods—Serum was obtained by venipuncture and stored at —20 C. Tears were induced by having the subject stare into a bright light or by placing several small crystals of sodium chloride in the lower conjunctiva. The tear samples (100
FEBRUARY, 1983
to 300 μΐ) were collected by glass capil lary tubes and stored at —70 C. Specific IgE antibodies to the pollen allergens were assayed in serum and tears by the radioallergosorbent test. Total IgE in serum and tear samples were quantitated by the paper radioimmunosorbent test. 7 We used transferrin, a serum pro tein with a molecular weight of approxi mately 70,000 daltons and which is not normally found in tears, as a marker for the leakage of serum proteins into the tear secretions. 9,10 Transferrin was mea sured by radial immunodiffusion using monospecific antisera. We quantitated IgG, IgM, and IgA in the tears by an enzyme-linked immunosorbent assay (ELISA), using a slightly modified version of the technique of Lindsten and associates. 11 Specific IgG antibodies to rye grass group I antigen and ragweed antigen E were measured by a double-antibody ELISA method. 12 Polystyrene microtiter plates were coated with rye grass group I antigen (20 μΐ, 5 μg/ml) or ragweed anti gen E (20 μΐ, 5 μg/ml) in a carbonatebicarbonate buffer (pH 9.6) (obtained from the National Institute of Allergy and Infectious Diseases). A positive control serum from a patient with hay fever who was undergoing pollen allergen desensitization was run on each plate to correct for daily variation. We used saliva from a nonatopic individual as a negative con trol. The absorbance was plotted against the log of the reciprocal serum or tear dilution. We quantitated specific anti body to rye grass group I antigen and ragweed antigen E by calculating the area under the serum or tear dilution curve (Fig. 1), and the results were expressed in arbitrary ELISA units. We calculated the local production of specific IgG antibody by the method of Mathews, 13 using the following formula: Index of local synthesis = (antibody in tears -r- transferrin in tears) — (antibody
VOL. 95, NO. 2
TEAR IGG ANTIBODIES IN CONJUNCTIVITIS
163
centrations of total IgG, IgM, IgA, and transferrin between the tear secretions and the serum. A ratio of more than 1.0 indicates local production. Because of wide differences in values between serum and tears, the immunoglobulin data were log transformed for analysis.
AREA-269 ELISA UNITS
o
RESULTS
Reciprocal Tear Dilution Fig. 1 (Ballow and associates). Tear dilution curve for IgG antibodies to rye grass group I antigen of a patient with vernal conjunctivitis. The area under the tear dilution curve between dilutions 1:2 and 1:32 was calculated and the results expressed as arbitrary ELISA units of specific IgG antibody.
in serum -s- transferrin in serum). This formula takes into account the variability and dilutional effects of sample collec tion. 13 An index of more than 0.2 repre sents appreciable local production of spe cific IgG antibody. This expression is derived from the formula of Donovan and associates. 14 We assessed the local pro duction of total IgG, IgM, and Ig A in tears by the method of Deuschl and Jo hansson, 15 using double ratios of the con
Mean serum IgE levels did not differ significantly among the various groups (Table 1). The mean total IgE in tears was significantly (P<.05) higher in patients with vernal conjunctivitis than in those with allergic conjunctivitis and the other control groups (Student's f-test). Patients with vernal conjunctivitis had significant ly (P<.01) more IgG (geometric mean = 91.5 μg/ml), and IgM (geometric mean = 10.8 μg/ml) in their tear secretions than did control subjects (Fig. 2). Of note, 26 of 30 patients with vernal conjunctivitis (87%) had measurable (IgM > 4 . 7 μg/ml) levels of IgM, whereas only three pa tients with allergic conjunctivitis and none of the control individuals had meas urable IgM levels in their tear secretions. The double ratio formula of Deuschl and Johansson 15 suggested that the tear IgG and IgM were locally produced, indicat ed by immunoglobulin-transferrin ratios of more than 1.0 (range, 6.3 to 249 for
TABLE 1 T O T A L I G E IN TEARS AND SERUM
Study Groups
Total IgE In tears (IU/ml) Geometric mean ± 1 S.D. In serum (IU/ml) Geometric mean ± 1 S.D.
Control (No. = 12)
Allergic Conjunctivitis (No. = 12)
Vernal Conjunctivitis (No. = 30)
1.1 <(0.25 to 1.4)
1.7 <(0.25 to 8.3)
3.7* <(0.25 to 25.3)
20.6 (3.0 to 143)
30.4 (5.8 to 160)
*P < . 0 5 by Student's t-test vs control levels.
49.7 (8.5 to 291)
164
AMERICAN JOURNAL OF OPHTHALMOLOGY
IgG
IgM
FEBRUARY, 1983
IgG and 2.8 to 186 for IgM). The tear IgA levels of patients with vernal conjunctivi tis did not differ from those of the other patient groups. Tear secretions were tested for the presence of IgG antibodies to the two inhalant pollen antigens: rye grass group I antigen 16 and antigen E of ragweed allergen. 17 Except for three individuals who had low amounts of IgG antibody to rye grass group I antigen (Fig. 3), the control group had few or no specific IgG antibodies to rye grass group I antigen (2.6 ELISA units) and ragweed antigen E (2.1 ELISA units) in their tears. Most patients with allergic conjunctivitis also had low amounts of specific IgG antibody to these two pollen antigens (for rye grass group I antigen, geometric mean = 4.5 ELISA units; for ragweed antigen E, geo metric mean = 6.4 ELISA units). In contrast, many of the patients with vernal conjunctivitis had substantial levels of specific IgG antibodies to rye grass group I antigen (geometric mean = 31.2;
IgA
Fig. 2 (Ballow and associates). Total immunoglobulins (IgG, IgM, and IgA) in the tears quantitated by ELISA. IgG and IgM were significantly (P<.001, Student's t-test) increased in the tear secretions of patients with vernal conjunctivitis (solid bars) com pared to control individuals (open bars) and patients with allergic conjunctivitis (slashed bars).
400
1 e
300
<
200
a
•
K
<
(9 a>
Fig. 3 (Ballow and associates). Tear IgG antibodies to rye grass group I antigen were quantitated by ELISA. The bars represent the geo metric mean ELISA units ±1 S.D. The tear levels of specific IgG anti bodies were significantly increased in the patients with vernal conjunc tivitis compared to those with aller gic conjunctivitis (P<.001, Student's t-test).
100 90 80 70 60 50 40 30
2
20
a
lOJr
Control
tKt AC
vc
L
VOL. 95, NO. 2
700 600 500 400
TEAR IGG ANTIBODIES IN CONJUNCTIVITIS
-
• •
-
•
300
3
I 3 3
<
200
100 90
165
-
Fig. 4 (Ballow and associates). Tear IgG specific antibodies to rag weed antigen E. The bars repre sent die geometric mean ELISA units ± 1 S.D. of antibody activity. Patients with vemal conjunctivitis had significantly (P<.05, Student's i-test) increased levels of IgG anti bodies compared to those with aller gic conjunctivitis.
• •
80 70 60
-
t
50
-
•
40 30
•
20
•
10.
··• .«fa. Control
+
t AC
—
• • ·· •• · VC
P<.001) and ragweed antigen E (geomet ric mean = 19.5; P<.05) in their tears (Figs. 3 and 4). Patients with vernal con junctivitis had a wide distribution in tear levels of IgG antibodies to these two pollen antigens. Table 2 shows the pro portion of patients with vernal and aller gic conjunctivitis who had tear levels of allergen-specific IgG antibodies more than 2 S.D. above the geometric mean of
control tear levels. Significantly more pa tients with vernal conjunctivitis than pa tients with allergic conjunctivitis had in creased allergen-specific IgG antibodies in their tears. Although 18 of 30 (60%) patients with vernal conjunctivitis had increased levels of IgG antibody to rye grass, this antibody was increased in only one of 12 patients with allergic conjuncti vitis (P<.001) (chi-square). Similarly, 20
TABLE 2 INCREASED LEVELS OF IGG ANTIBODIES IN TEARS*
Study Group Antigen Rye grass No. positive Geometic mean (ELISA units) Ragweed antigen E No. positive Geometric mean (ELISA units)
Vernal Conjunctivitis
Allergic Conjunctivitis
18 of 30
l o f 12 <.001
68.7 20 of 30
4 of 12
50.0
20.5
*More than 2 S.D. from geometric mean for control group. By chi square analysis.
+
P+
<.05
166
AMERICAN JOURNAL OF OPHTHALMOLOGY
of 30 (67%) patients with vernal conjunc tivitis but only four of 12 (33%) with allergic conjunctivitis had increased IgG antibody levels to ragweed antigen E (P<.05). We used the formulas of Mathews 13 and Donovan and associates 14 to estimate the local conjunctival contribution of specific IgG antibody in the tear secretions. Transferrin was used as a marker for the transudation of antibodies from the serum into the tears. Fourteen patients with vernal conjunctivitis for whom ade quate tear samples were available for testing had local production of IgG specif ic antibodies for ragweed antigen E. The local production ranged from 20% to 99.6% (mean, 81%). Similarly, for rye grass group I antigen, in 16 patients tested most (29% to 99.9%; mean, 83.5%) of the specific IgG antibodies in the tear secretions were locally produced by the conjunctival tissues. The index of local synthesis also indicated that the rye grass (mean, 3.7; range, 0.06 to 18.6) and rag weed antigen E (mean, 2.87; range, 0.04 to 9.94) IgG antibodies were locally pro duced in many of the tear samples. Of the 30 patients with vernal conjunc tivitis, 23 (76.7%) had IgG antibodies in their tears to rye grass antigen or rag weed antigen E, or both, at levels more than 2 S.D. from the geometric mean of control tears, whereas only 13 (43%) had pollen-specific IgE antibodies in their tears. Of the 17 patients with vernal conjunctivitis who had no pollen-specific IgE antibodies in their tears, 14 (82.4%) had IgG antibodies in their tears to rye grass or ragweed antigen E or both. Con versely, nine of the 13 (69.2%) who had detectable tear pollen-specific IgE anti bodies had IgG antibodies to rye grass or antigen E or both. Although the patients with vernal conjunctivitis but without pollen-specific IgE antibodies in their tears tended to have a higher frequency of specific tear IgG antibodies (82.4%) to
FEBRUARY, 1983
rye grass or antigen E or both in compari son to those patients who had detectable IgE antibodies (69.2%), this difference did not reach significance (chi-square). DISCUSSION
Our initial studies showed that specific IgE antibodies to the inhalant pollen al lergens could be found in the tears of patients with vernal conjunctivitis. 7 These findings complemented the previ ous work of Allansmith, Hahn, and Simon, 2 Brauninger and Centifanto, 18 and Little, Centifanto, and Kaufman, 19 whose investigations of immunoglobulin levels in tear secretions suggested that vernal conjunctivitis was an IgE-mediated disor der. The presence of increased histamine levels in tears 20 and the increased num bers of IgE plasma cells, mast cells, basophils, and eosinophils in the conjunctival tissues 3 of patients with vernal conjuncti vitis provided further support for an IgEmediated pathogenesis. Evidence that suggests a non-IgEmediated immune mechanism for the pathogenesis of vernal conjunctivitis in cludes the mixed cellular infiltrate in the conjunctival tissues, 3 ' 6,8 the often poor clinical response to topical antihistamines and decongestant therapy, the uniformly good response to corticosteroid therapy, and the absence of an atopic diathesis in many patients living in the more north ern latitudes of North America (K. Mathews, oral communication, May 1982). Our study showed that the tears of patients with vernal conjunctivitis con tain specific IgG antibodies to rye grass group I antigen or ragweed antigen E pollen antigens or both. Normal nonatopic individuals and individuals with seasonal rhinitis had few or no specific IgG antibodies to these two pollen anti gens in their tear secretions. A few of the patients with allergic conjunctivitis had specific IgG antibodies in their tears but
VOL. 95, NO. 2
TEAR IGG ANTIBODIES IN CONJUNCTIVITIS
the levels were far below the amounts seen in the tears of patients with vernal conjunctivitis (Figs. 3 and 4). Additional ly, using transferrin as a marker for the leakage of plasma proteins into tears and the formulas of Mathews 13 and Donovan and associates 14 demonstrated that a large proportion of the specific IgG antibodies were locally produced by the conjunctival tissues. Four patients had higher levels of specific IgG antibodies to either rye grass or ragweed antigen E in their tears than in their sera. These results, along with our earlier findings that IgE antibodies were present in the tears but not the sera of six patients with vernal conjunctivitis, 7 strongly supported the hypothesis that the external eye can produce a local im mune response even in the absence of systemic immunity. The levels of total IgE, IgG, and IgM, as well as specific IgE and IgG, were also increased in the tear secretions of our patients with vernal conjunctivitis. McClellan and associates21 demonstrated sim ilar increases in tear immunoglobulin lev els in their patients with vernal conjunctivitis. Tear IgM was of consider able interest in that 26 of 30 patients with vernal conjunctivitis (87%) had increased amounts of IgM. Allansmith (oral commu nication, March 1982) suggested that the quantitative tear immunoglobulins, that is, increased IgM, and the presence of basophils in the conjunctival tissues, are helpful diagnostic criteria for vernal con junctivitis in the presence of a suggestive history and physical findings. A study of the relationship between pollen-specific IgE antibodies and the presence of specific IgG antibodies to rye grass or ragweed antigen E, or both, in the tears showed that fewer patients with vernal conjunctivitis and tear IgE anti bodies had specific tear IgG antibodies (69.2%) than patients (82.4%) with undetectable pollen-specific IgE antibodies. How these specific IgG antibodies relate
167
to the pathophysiology of vernal conjunc tivitis is not known. Classically, in aller gic diseases, IgG antibodies have served as blocking antibodies for IgE-mediated immune reactions. 22 However, an IgGmediated and an IgE-mediated immuno logie pathogenesis for vernal conjunctivi tis are not mutually exclusive. For example, the specific IgG antibodies may participate in an immune complex immu nologie reaction with activation of com plement proteins. Several complement proteins have been found in tears includ ing C4, 23 C3, 24 and factor B of the alterna tive pathway. 25 Activation of complement can result in changes in capillary perme ability and the release of vasoactive am ines from basophils and mast cells. 26 Finally, there is evidence that the IgG4 subclass of IgG can act as a reaginic immunoglobulin, and thus mimic IgEmediated disease. 27 Preliminary studies in our laboratory have demonstrated that the IgG antibodies to rye grass and rag weed antigen E, at least in part, belong to the IgG4 subclass. Further studies are necessary to understand the clinical sig nificance of these IgG antibodies and their role in the pathogenesis of vernal conjunctivitis. REFERENCES 1. Allansmith, M., and Frick, O. L.: Antibodies to grass in vernal conjunctivitis. J. Allergy 34:535, 1963. 2. Allansmith, M. R., Hahn, G. S., and Simon, M. A.: Tissue, tear, and serum IgE concentrations in vernal conjunctivitis. Am. J. Ophthalmol. 81:506, 1976. 3. Allansmith, M. R., and Baird, R. S.: Mast cells, eosinophils and basophils in vernal conjunctivitis. J. Allergy Clin. Immunol. 61:154, 1978. 4. Bloch-Michel, E., Audoin-Berault, J., Diebold, J., Herman, D., Dry, J., and Campinchi, R. : Etude en immunofluorescence des plasmocytes de la con jonctive allergique en particulier des cellules forma trices des immunoglobulines E (IgE). Arch. Ophtalmol. 37:89, 1977. 5. Rice, N. S. C , and Jones, B. R. : Vernal keratoconjunctivitis. An allergie disease of the eyes of children. In Brostoff, J. (ed.): Clinical Immunology and Allergy in Pediatrie Medicine. Oxford, Blackweil Scientific Publishers, 1973, pp. 139-147.
168
AMERICAN JOURNAL OF OPHTHALMOLOGY
6. Easty, D. L., Birkinshaw, M., Merrett, T., Merrett, J., and Madden, P.: Immunology of vernal disease. In Pepys, J., and Edwards, A. M. (eds.): The Mast Cell. Its Role in Health and Disease. Switzer land, Pitman Medical, 1979, pp. 493-502. 7. Ballow, M., and Mendelson, L. : Specific immunoglobulin E antibodies in tear secretions of patients with vernal conjunctivitis. J. Allergy Clin. Immunol. 66:112, 1980. 8. Morgan, G.: The pathology of vernal conjuncti vitis. Trans. Ophthalmol. Soc. U.K. 91:467, 1971. 9. Josephson, A. S., andWeiner, R. A.: Studies of the proteins of lacrimai secretions. J. Immunol. 100:1080, 1968. 10. Van Haeringen, N. J.: Clinical biochemistry of tears. Surv. Ophthalmol. 26:84, 1981. 11. Lindsten, T., Seeley, J. K., Ballow, M., Saka moto, K., St. Onge, S., Yetz, J., Aman, P., and Purtilo, D. T.: Immune deficiency in the X-linked lymphoproliferative syndrome. II. Immunoregulatory T cell defects. J. Immunol., in press. 12. Sepulveda, R., Longbottom, J. L., and Pepys, J.: Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens of Aspergillus fumigatus. Clin. Allergy 9:359, 1979. 13. Mathews, K. P. : Calculation of secretory anti bodies and immunoglobulins. J. Allergy Clin. Immunol. 68:46, 1981. 14. Donovan, R., Johansson, S. G. O., Bennich, H., and Soothill, J. F.: Immunoglobulins in nasal polyp fluid. Int. Arch. Allergy Appi. Immunol. 37:154, 1970. 15. Deuschl, H., and Johansson, S. G. O.: Immu noglobulins in tracheobronchial secretion with spe cial reference to IgE. Clin. Exp. Immunol. 16:401, 1974. 16. Lieferman, K. M., and Gleich, G. J.: The cross-reactivity of IgE antibodies with pollen aller gens. I. Analyses of various species of grass pollens. J. Allergy Clin. Immunol. 58:129, 1976. 17. Pauli, B. R., Gleich, G. J., and Atassi, M. Z.:
FEBRUARY, 1983
Structure and activity of ragweed antigen E. III. The role of the thiol group in the conformation and allergenic activity of antigen E. Mol. Immunol. 17:281, 1980. 18. Brauninger, G. E., and Centifanto, Y. M.: Immunoglobulin E in human tears. Am. J. Ophthal mol. 72:558, 1971. 19. Little, J. M., Centifanto, Y., and Kaufman, H. E.: Immunoglobulins in tears. Am. J. Ophthal mol. 68:898, 1969. 20. Abelson, M. B., Soter, N. A., Simon, M. A., Dohlman, J., and Allansmith, M. R.: Histamine in human tears. Am. J. Ophthalmol. 83:417, 1977. 21. McClellan, B. H., Whitney, C. R., Newman, L. P., and Allansmith, M. R.: Immunoglobulins in tears. Am. J. Ophthalmol. 76:89, 1973. 22. Lichtenstein, L. M., Holtzman, N. A., and Burnett, L. S.: A quantitative in vitro study of the Chromatographie distribution and immunoglobulin characteristics of human blocking antibody. J. Im munol. 101:317, 1968. 23. Chandler, J. W., Leder, R., Kaufman, H. E., and Caldwell, J. R. : Quantitative determinations of complement components and immunoglobulins in tears and aqueous humor. Invest. Ophthalmol. 13:151, 1974. 24. Bluestone, R., Easty, D. L., Goldberg, L. S., Jones, B. R., and Pettit, T. H.: Lacrimai immuno globulins and complement quantified by counterimmunoelectrophoresis. Br. J. Ophthalmol. 59:279, 1975. 25. Yamamoto, G. K., and Allansmith, M. R.: Complement in tears from normal humans. Am. J. Ophthalmol. 88:758, 1979. 26. Johnson, A. R., Hugli, T. E., and MullerEberhard, H. J.: Release of histamine from mast cells by the complement peptides C3A and C5A. Immunology 28:1067, 1975. 27. Gwynn, C. M., Ingram, J., Almousawi, T., and Stanworth, D. R. : Bronchial provocation tests in atopic patients with allergen-specific IgG4 antibodies. Lancet 1:254, 1982.
M.D.,
We studied the tears of 30 patients (25 males and five females, ranging in age from 8 to 34 years) with vernal conjunctivitis for pollen-specific IgG antibodies to rye grass and ragweed antigen E by an enzyme-linked immunosorbent assay (ELISA). Eighteen of 30 (60%) and 20 of 30 (67%) patients with vernal conjunctivitis had significant levels (more than 2 S.D. from the mean of control tears) of IgG antibodies in their tear secretions to rye grass (geometric mean = 68.7 ELISA units) and ragweed antigen E (geometric mean = 50 ELISA units), respective ly. In contrast, the control groups (eight atopic individuals, four with seasonal rhinitis, and 12 with allergic conjunctivitis) had low amounts of specific IgG antibodies to these two pollen antigens in their tears. Total IgG and IgM were also increased in the tears of patients with vernal conjunctivitis. To evaluate whether these immunoglobulins and specific IgG antibodies were locally produced by the conjunctival tissues, we used transferrin as a marker for the leakage of plasma proteins into tears. We found that the specific IgG antibodies to rye grass or ragweed antigen E, or both, in the tears were locally produced by the conjuncti val tissues. The local production ranged from 20% to 99.9%. Of 17 patients with vernal conjunctivitis and undetectable pollen-specific IgE antibodies in their tears, 14 (82.4%) had tear specific IgG antibodies to rye grass or ragweed antigen E or both, whereas of those with measurable tear IgE antibodies, only nine of 13 (69%) had tear IgG antibodies to rye grass or antigen E or both. These results suggested that both IgE- and IgG-mediated immune mechanisms may be impor tant in the pathogenesis of vernal conjunctivitis. Vernal conjunctivitis, a bilateral, often severe inflammatory condition of the con junctiva, is characterized by itching, tearAccepted for publication Oct. 25, 1982. From the Departments of Pediatrics (Drs. Ballow and Mendelson, Mrs. Rapacz, and Mr. Sparks) and Ophthalmology (Dr. Donshik), University of Connec ticut Health Center School of Medicine, Farmington, Connecticut. This study was supported by grant EY-02036 from the National Eye Institute (Dr. Ballow). Reprint requests to Mark Ballow, M.D., Depart ment of Pediatrics, University of Connecticut Health Center, Farmington, CT 06032.
ing, and photophobia. Papillary hypertro phy of the upper tarsal conjunctiva and the presence of eosinophils in tear secre tions are pathognomonic of this condi tion. These clinical manifestations and the studies by Allansmith and associates1"3 and others4*6 suggest that vernal conjunc tivitis is an atopic ocular disorder. Fur ther support for an IgE-mediated patho genesis was recently reported in studies from our laboratory in which specific IgE antibodies to pollen allergens were dem onstrated in the tear secretions of more
©AMERICAN JOURNAL OF OPHTHALMOLOGY 95:161-168, 1983
161
162
AMERICAN JOURNAL OF OPHTHALMOLOGY
than one half the patients with vernal conjunctivitis studied. 7 However, the complex histopathology 3,6,8 suggests that other immune mechanisms may play a role in the pathogenesis of vernal con junctivitis. We measured specific IgG antibodies in the tears to two inhalant pollen aller gens, rye grass and ragweed antigen E, in patients with vernal conjunctivitis and in control subjects. We also investigated whether these specific IgG antibodies were locally produced by the conjunctival tissues of the eye. Our finding of locally produced pollen-specific IgG antibody together with our previous studies 7 sug gested that both IgE- and IgG-mediated immune mechanisms are involved in the pathogenesis of vernal conjunctivitis. S U B J E C T S AND M E T H O D S
Thirty patients (25 males and five fe males) with vernal conjunctivitis had the typical symptoms and physical findings of vernal conjunctivitis and had had their disease two or more seasons. The patients ranged in age from 8 to 34 years (mean, 13 years). Thirteen patients also had other allergic disorders, mostly hay fever and eczema. Only seven of 24 patients who underwent skin testing for immedi ate hypersensitivity had positive skin re activity to inhalant pollen allergens. Control groups included eight nonatopic individuals, four individuals with seasonal rhinitis, and 12 patients with allergic conjunctivitis. Tear samples were obtained approximately three to four weeks after peak symptomatology and at least two weeks after topical corticosteroids had been discontinued. Methods—Serum was obtained by venipuncture and stored at —20 C. Tears were induced by having the subject stare into a bright light or by placing several small crystals of sodium chloride in the lower conjunctiva. The tear samples (100
FEBRUARY, 1983
to 300 μΐ) were collected by glass capil lary tubes and stored at —70 C. Specific IgE antibodies to the pollen allergens were assayed in serum and tears by the radioallergosorbent test. Total IgE in serum and tear samples were quantitated by the paper radioimmunosorbent test. 7 We used transferrin, a serum pro tein with a molecular weight of approxi mately 70,000 daltons and which is not normally found in tears, as a marker for the leakage of serum proteins into the tear secretions. 9,10 Transferrin was mea sured by radial immunodiffusion using monospecific antisera. We quantitated IgG, IgM, and IgA in the tears by an enzyme-linked immunosorbent assay (ELISA), using a slightly modified version of the technique of Lindsten and associates. 11 Specific IgG antibodies to rye grass group I antigen and ragweed antigen E were measured by a double-antibody ELISA method. 12 Polystyrene microtiter plates were coated with rye grass group I antigen (20 μΐ, 5 μg/ml) or ragweed anti gen E (20 μΐ, 5 μg/ml) in a carbonatebicarbonate buffer (pH 9.6) (obtained from the National Institute of Allergy and Infectious Diseases). A positive control serum from a patient with hay fever who was undergoing pollen allergen desensitization was run on each plate to correct for daily variation. We used saliva from a nonatopic individual as a negative con trol. The absorbance was plotted against the log of the reciprocal serum or tear dilution. We quantitated specific anti body to rye grass group I antigen and ragweed antigen E by calculating the area under the serum or tear dilution curve (Fig. 1), and the results were expressed in arbitrary ELISA units. We calculated the local production of specific IgG antibody by the method of Mathews, 13 using the following formula: Index of local synthesis = (antibody in tears -r- transferrin in tears) — (antibody
VOL. 95, NO. 2
TEAR IGG ANTIBODIES IN CONJUNCTIVITIS
163
centrations of total IgG, IgM, IgA, and transferrin between the tear secretions and the serum. A ratio of more than 1.0 indicates local production. Because of wide differences in values between serum and tears, the immunoglobulin data were log transformed for analysis.
AREA-269 ELISA UNITS
o
RESULTS
Reciprocal Tear Dilution Fig. 1 (Ballow and associates). Tear dilution curve for IgG antibodies to rye grass group I antigen of a patient with vernal conjunctivitis. The area under the tear dilution curve between dilutions 1:2 and 1:32 was calculated and the results expressed as arbitrary ELISA units of specific IgG antibody.
in serum -s- transferrin in serum). This formula takes into account the variability and dilutional effects of sample collec tion. 13 An index of more than 0.2 repre sents appreciable local production of spe cific IgG antibody. This expression is derived from the formula of Donovan and associates. 14 We assessed the local pro duction of total IgG, IgM, and Ig A in tears by the method of Deuschl and Jo hansson, 15 using double ratios of the con
Mean serum IgE levels did not differ significantly among the various groups (Table 1). The mean total IgE in tears was significantly (P<.05) higher in patients with vernal conjunctivitis than in those with allergic conjunctivitis and the other control groups (Student's f-test). Patients with vernal conjunctivitis had significant ly (P<.01) more IgG (geometric mean = 91.5 μg/ml), and IgM (geometric mean = 10.8 μg/ml) in their tear secretions than did control subjects (Fig. 2). Of note, 26 of 30 patients with vernal conjunctivitis (87%) had measurable (IgM > 4 . 7 μg/ml) levels of IgM, whereas only three pa tients with allergic conjunctivitis and none of the control individuals had meas urable IgM levels in their tear secretions. The double ratio formula of Deuschl and Johansson 15 suggested that the tear IgG and IgM were locally produced, indicat ed by immunoglobulin-transferrin ratios of more than 1.0 (range, 6.3 to 249 for
TABLE 1 T O T A L I G E IN TEARS AND SERUM
Study Groups
Total IgE In tears (IU/ml) Geometric mean ± 1 S.D. In serum (IU/ml) Geometric mean ± 1 S.D.
Control (No. = 12)
Allergic Conjunctivitis (No. = 12)
Vernal Conjunctivitis (No. = 30)
1.1 <(0.25 to 1.4)
1.7 <(0.25 to 8.3)
3.7* <(0.25 to 25.3)
20.6 (3.0 to 143)
30.4 (5.8 to 160)
*P < . 0 5 by Student's t-test vs control levels.
49.7 (8.5 to 291)
164
AMERICAN JOURNAL OF OPHTHALMOLOGY
IgG
IgM
FEBRUARY, 1983
IgG and 2.8 to 186 for IgM). The tear IgA levels of patients with vernal conjunctivi tis did not differ from those of the other patient groups. Tear secretions were tested for the presence of IgG antibodies to the two inhalant pollen antigens: rye grass group I antigen 16 and antigen E of ragweed allergen. 17 Except for three individuals who had low amounts of IgG antibody to rye grass group I antigen (Fig. 3), the control group had few or no specific IgG antibodies to rye grass group I antigen (2.6 ELISA units) and ragweed antigen E (2.1 ELISA units) in their tears. Most patients with allergic conjunctivitis also had low amounts of specific IgG antibody to these two pollen antigens (for rye grass group I antigen, geometric mean = 4.5 ELISA units; for ragweed antigen E, geo metric mean = 6.4 ELISA units). In contrast, many of the patients with vernal conjunctivitis had substantial levels of specific IgG antibodies to rye grass group I antigen (geometric mean = 31.2;
IgA
Fig. 2 (Ballow and associates). Total immunoglobulins (IgG, IgM, and IgA) in the tears quantitated by ELISA. IgG and IgM were significantly (P<.001, Student's t-test) increased in the tear secretions of patients with vernal conjunctivitis (solid bars) com pared to control individuals (open bars) and patients with allergic conjunctivitis (slashed bars).
400
1 e
300
<
200
a
•
K
<
(9 a>
Fig. 3 (Ballow and associates). Tear IgG antibodies to rye grass group I antigen were quantitated by ELISA. The bars represent the geo metric mean ELISA units ±1 S.D. The tear levels of specific IgG anti bodies were significantly increased in the patients with vernal conjunc tivitis compared to those with aller gic conjunctivitis (P<.001, Student's t-test).
100 90 80 70 60 50 40 30
2
20
a
lOJr
Control
tKt AC
vc
L
VOL. 95, NO. 2
700 600 500 400
TEAR IGG ANTIBODIES IN CONJUNCTIVITIS
-
• •
-
•
300
3
I 3 3
<
200
100 90
165
-
Fig. 4 (Ballow and associates). Tear IgG specific antibodies to rag weed antigen E. The bars repre sent die geometric mean ELISA units ± 1 S.D. of antibody activity. Patients with vemal conjunctivitis had significantly (P<.05, Student's i-test) increased levels of IgG anti bodies compared to those with aller gic conjunctivitis.
• •
80 70 60
-
t
50
-
•
40 30
•
20
•
10.
··• .«fa. Control
+
t AC
—
• • ·· •• · VC
P<.001) and ragweed antigen E (geomet ric mean = 19.5; P<.05) in their tears (Figs. 3 and 4). Patients with vernal con junctivitis had a wide distribution in tear levels of IgG antibodies to these two pollen antigens. Table 2 shows the pro portion of patients with vernal and aller gic conjunctivitis who had tear levels of allergen-specific IgG antibodies more than 2 S.D. above the geometric mean of
control tear levels. Significantly more pa tients with vernal conjunctivitis than pa tients with allergic conjunctivitis had in creased allergen-specific IgG antibodies in their tears. Although 18 of 30 (60%) patients with vernal conjunctivitis had increased levels of IgG antibody to rye grass, this antibody was increased in only one of 12 patients with allergic conjuncti vitis (P<.001) (chi-square). Similarly, 20
TABLE 2 INCREASED LEVELS OF IGG ANTIBODIES IN TEARS*
Study Group Antigen Rye grass No. positive Geometic mean (ELISA units) Ragweed antigen E No. positive Geometric mean (ELISA units)
Vernal Conjunctivitis
Allergic Conjunctivitis
18 of 30
l o f 12 <.001
68.7 20 of 30
4 of 12
50.0
20.5
*More than 2 S.D. from geometric mean for control group. By chi square analysis.
+
P+
<.05
166
AMERICAN JOURNAL OF OPHTHALMOLOGY
of 30 (67%) patients with vernal conjunc tivitis but only four of 12 (33%) with allergic conjunctivitis had increased IgG antibody levels to ragweed antigen E (P<.05). We used the formulas of Mathews 13 and Donovan and associates 14 to estimate the local conjunctival contribution of specific IgG antibody in the tear secretions. Transferrin was used as a marker for the transudation of antibodies from the serum into the tears. Fourteen patients with vernal conjunctivitis for whom ade quate tear samples were available for testing had local production of IgG specif ic antibodies for ragweed antigen E. The local production ranged from 20% to 99.6% (mean, 81%). Similarly, for rye grass group I antigen, in 16 patients tested most (29% to 99.9%; mean, 83.5%) of the specific IgG antibodies in the tear secretions were locally produced by the conjunctival tissues. The index of local synthesis also indicated that the rye grass (mean, 3.7; range, 0.06 to 18.6) and rag weed antigen E (mean, 2.87; range, 0.04 to 9.94) IgG antibodies were locally pro duced in many of the tear samples. Of the 30 patients with vernal conjunc tivitis, 23 (76.7%) had IgG antibodies in their tears to rye grass antigen or rag weed antigen E, or both, at levels more than 2 S.D. from the geometric mean of control tears, whereas only 13 (43%) had pollen-specific IgE antibodies in their tears. Of the 17 patients with vernal conjunctivitis who had no pollen-specific IgE antibodies in their tears, 14 (82.4%) had IgG antibodies in their tears to rye grass or ragweed antigen E or both. Con versely, nine of the 13 (69.2%) who had detectable tear pollen-specific IgE anti bodies had IgG antibodies to rye grass or antigen E or both. Although the patients with vernal conjunctivitis but without pollen-specific IgE antibodies in their tears tended to have a higher frequency of specific tear IgG antibodies (82.4%) to
FEBRUARY, 1983
rye grass or antigen E or both in compari son to those patients who had detectable IgE antibodies (69.2%), this difference did not reach significance (chi-square). DISCUSSION
Our initial studies showed that specific IgE antibodies to the inhalant pollen al lergens could be found in the tears of patients with vernal conjunctivitis. 7 These findings complemented the previ ous work of Allansmith, Hahn, and Simon, 2 Brauninger and Centifanto, 18 and Little, Centifanto, and Kaufman, 19 whose investigations of immunoglobulin levels in tear secretions suggested that vernal conjunctivitis was an IgE-mediated disor der. The presence of increased histamine levels in tears 20 and the increased num bers of IgE plasma cells, mast cells, basophils, and eosinophils in the conjunctival tissues 3 of patients with vernal conjuncti vitis provided further support for an IgEmediated pathogenesis. Evidence that suggests a non-IgEmediated immune mechanism for the pathogenesis of vernal conjunctivitis in cludes the mixed cellular infiltrate in the conjunctival tissues, 3 ' 6,8 the often poor clinical response to topical antihistamines and decongestant therapy, the uniformly good response to corticosteroid therapy, and the absence of an atopic diathesis in many patients living in the more north ern latitudes of North America (K. Mathews, oral communication, May 1982). Our study showed that the tears of patients with vernal conjunctivitis con tain specific IgG antibodies to rye grass group I antigen or ragweed antigen E pollen antigens or both. Normal nonatopic individuals and individuals with seasonal rhinitis had few or no specific IgG antibodies to these two pollen anti gens in their tear secretions. A few of the patients with allergic conjunctivitis had specific IgG antibodies in their tears but
VOL. 95, NO. 2
TEAR IGG ANTIBODIES IN CONJUNCTIVITIS
the levels were far below the amounts seen in the tears of patients with vernal conjunctivitis (Figs. 3 and 4). Additional ly, using transferrin as a marker for the leakage of plasma proteins into tears and the formulas of Mathews 13 and Donovan and associates 14 demonstrated that a large proportion of the specific IgG antibodies were locally produced by the conjunctival tissues. Four patients had higher levels of specific IgG antibodies to either rye grass or ragweed antigen E in their tears than in their sera. These results, along with our earlier findings that IgE antibodies were present in the tears but not the sera of six patients with vernal conjunctivitis, 7 strongly supported the hypothesis that the external eye can produce a local im mune response even in the absence of systemic immunity. The levels of total IgE, IgG, and IgM, as well as specific IgE and IgG, were also increased in the tear secretions of our patients with vernal conjunctivitis. McClellan and associates21 demonstrated sim ilar increases in tear immunoglobulin lev els in their patients with vernal conjunctivitis. Tear IgM was of consider able interest in that 26 of 30 patients with vernal conjunctivitis (87%) had increased amounts of IgM. Allansmith (oral commu nication, March 1982) suggested that the quantitative tear immunoglobulins, that is, increased IgM, and the presence of basophils in the conjunctival tissues, are helpful diagnostic criteria for vernal con junctivitis in the presence of a suggestive history and physical findings. A study of the relationship between pollen-specific IgE antibodies and the presence of specific IgG antibodies to rye grass or ragweed antigen E, or both, in the tears showed that fewer patients with vernal conjunctivitis and tear IgE anti bodies had specific tear IgG antibodies (69.2%) than patients (82.4%) with undetectable pollen-specific IgE antibodies. How these specific IgG antibodies relate
167
to the pathophysiology of vernal conjunc tivitis is not known. Classically, in aller gic diseases, IgG antibodies have served as blocking antibodies for IgE-mediated immune reactions. 22 However, an IgGmediated and an IgE-mediated immuno logie pathogenesis for vernal conjunctivi tis are not mutually exclusive. For example, the specific IgG antibodies may participate in an immune complex immu nologie reaction with activation of com plement proteins. Several complement proteins have been found in tears includ ing C4, 23 C3, 24 and factor B of the alterna tive pathway. 25 Activation of complement can result in changes in capillary perme ability and the release of vasoactive am ines from basophils and mast cells. 26 Finally, there is evidence that the IgG4 subclass of IgG can act as a reaginic immunoglobulin, and thus mimic IgEmediated disease. 27 Preliminary studies in our laboratory have demonstrated that the IgG antibodies to rye grass and rag weed antigen E, at least in part, belong to the IgG4 subclass. Further studies are necessary to understand the clinical sig nificance of these IgG antibodies and their role in the pathogenesis of vernal conjunctivitis. REFERENCES 1. Allansmith, M., and Frick, O. L.: Antibodies to grass in vernal conjunctivitis. J. Allergy 34:535, 1963. 2. Allansmith, M. R., Hahn, G. S., and Simon, M. A.: Tissue, tear, and serum IgE concentrations in vernal conjunctivitis. Am. J. Ophthalmol. 81:506, 1976. 3. Allansmith, M. R., and Baird, R. S.: Mast cells, eosinophils and basophils in vernal conjunctivitis. J. Allergy Clin. Immunol. 61:154, 1978. 4. Bloch-Michel, E., Audoin-Berault, J., Diebold, J., Herman, D., Dry, J., and Campinchi, R. : Etude en immunofluorescence des plasmocytes de la con jonctive allergique en particulier des cellules forma trices des immunoglobulines E (IgE). Arch. Ophtalmol. 37:89, 1977. 5. Rice, N. S. C , and Jones, B. R. : Vernal keratoconjunctivitis. An allergie disease of the eyes of children. In Brostoff, J. (ed.): Clinical Immunology and Allergy in Pediatrie Medicine. Oxford, Blackweil Scientific Publishers, 1973, pp. 139-147.
168
AMERICAN JOURNAL OF OPHTHALMOLOGY
6. Easty, D. L., Birkinshaw, M., Merrett, T., Merrett, J., and Madden, P.: Immunology of vernal disease. In Pepys, J., and Edwards, A. M. (eds.): The Mast Cell. Its Role in Health and Disease. Switzer land, Pitman Medical, 1979, pp. 493-502. 7. Ballow, M., and Mendelson, L. : Specific immunoglobulin E antibodies in tear secretions of patients with vernal conjunctivitis. J. Allergy Clin. Immunol. 66:112, 1980. 8. Morgan, G.: The pathology of vernal conjuncti vitis. Trans. Ophthalmol. Soc. U.K. 91:467, 1971. 9. Josephson, A. S., andWeiner, R. A.: Studies of the proteins of lacrimai secretions. J. Immunol. 100:1080, 1968. 10. Van Haeringen, N. J.: Clinical biochemistry of tears. Surv. Ophthalmol. 26:84, 1981. 11. Lindsten, T., Seeley, J. K., Ballow, M., Saka moto, K., St. Onge, S., Yetz, J., Aman, P., and Purtilo, D. T.: Immune deficiency in the X-linked lymphoproliferative syndrome. II. Immunoregulatory T cell defects. J. Immunol., in press. 12. Sepulveda, R., Longbottom, J. L., and Pepys, J.: Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens of Aspergillus fumigatus. Clin. Allergy 9:359, 1979. 13. Mathews, K. P. : Calculation of secretory anti bodies and immunoglobulins. J. Allergy Clin. Immunol. 68:46, 1981. 14. Donovan, R., Johansson, S. G. O., Bennich, H., and Soothill, J. F.: Immunoglobulins in nasal polyp fluid. Int. Arch. Allergy Appi. Immunol. 37:154, 1970. 15. Deuschl, H., and Johansson, S. G. O.: Immu noglobulins in tracheobronchial secretion with spe cial reference to IgE. Clin. Exp. Immunol. 16:401, 1974. 16. Lieferman, K. M., and Gleich, G. J.: The cross-reactivity of IgE antibodies with pollen aller gens. I. Analyses of various species of grass pollens. J. Allergy Clin. Immunol. 58:129, 1976. 17. Pauli, B. R., Gleich, G. J., and Atassi, M. Z.:
FEBRUARY, 1983
Structure and activity of ragweed antigen E. III. The role of the thiol group in the conformation and allergenic activity of antigen E. Mol. Immunol. 17:281, 1980. 18. Brauninger, G. E., and Centifanto, Y. M.: Immunoglobulin E in human tears. Am. J. Ophthal mol. 72:558, 1971. 19. Little, J. M., Centifanto, Y., and Kaufman, H. E.: Immunoglobulins in tears. Am. J. Ophthal mol. 68:898, 1969. 20. Abelson, M. B., Soter, N. A., Simon, M. A., Dohlman, J., and Allansmith, M. R.: Histamine in human tears. Am. J. Ophthalmol. 83:417, 1977. 21. McClellan, B. H., Whitney, C. R., Newman, L. P., and Allansmith, M. R.: Immunoglobulins in tears. Am. J. Ophthalmol. 76:89, 1973. 22. Lichtenstein, L. M., Holtzman, N. A., and Burnett, L. S.: A quantitative in vitro study of the Chromatographie distribution and immunoglobulin characteristics of human blocking antibody. J. Im munol. 101:317, 1968. 23. Chandler, J. W., Leder, R., Kaufman, H. E., and Caldwell, J. R. : Quantitative determinations of complement components and immunoglobulins in tears and aqueous humor. Invest. Ophthalmol. 13:151, 1974. 24. Bluestone, R., Easty, D. L., Goldberg, L. S., Jones, B. R., and Pettit, T. H.: Lacrimai immuno globulins and complement quantified by counterimmunoelectrophoresis. Br. J. Ophthalmol. 59:279, 1975. 25. Yamamoto, G. K., and Allansmith, M. R.: Complement in tears from normal humans. Am. J. Ophthalmol. 88:758, 1979. 26. Johnson, A. R., Hugli, T. E., and MullerEberhard, H. J.: Release of histamine from mast cells by the complement peptides C3A and C5A. Immunology 28:1067, 1975. 27. Gwynn, C. M., Ingram, J., Almousawi, T., and Stanworth, D. R. : Bronchial provocation tests in atopic patients with allergen-specific IgG4 antibodies. Lancet 1:254, 1982.