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T and B Lymphocytes in Ocular Cicatricial Pemphigoid
T A N D B LYMPHOCYTES IN O C U L A R CICATRICIAL P E M P H I G O I D B A R T L Y J. M O N D I N O , M . D . ,
H I A H W A R A O , M.S.,
A N D S T U A R T I. B R O W N ,
M.D.
Pittsburgh, Pennsylvania
We performed peripheral blood T and B lymphocyte enumerations on 36 patients with ocular cicatricial pemphigoid and 49 age-matched controls. We found a decrease in both the mean percentage and the absolute number of T lymphocytes (determined by E rosettes) in patients compared to controls. We also found a decrease in the mean percentages and absolute numbers of B lymphocytes (determined by EAC and EA rosettes) in patients compared to controls.
Cicatricial pemphigoid is a chronic dis ease characterized by recurrent blisters or bullae of the skin and mucous mem branes and a tendency for scar forma tion. 1 Characteristic features of ocular involvement include progressive shrink age of the conjunctiva, entropion, trichiasis, xerosis, and, ultimately, reduced vision from corneal opacification. Im munologie abnormalities are associated with this disease. In cicatricial pemphi goid, immunoglobulins and components of both the alternative and classical com plement pathways are bound to the base ment membrane zone of skin and oral mucosa. 2,3 Immunopathologic studies of the conjunctiva showed immunoglobulins and the third component of complement bound not only to the conjunctival base ment membrane but also to its epitheli um. 4 ' 5 Circulating antibodies that bind to the basement membrane zone of skin and oral mucosa have occasionally been dem onstrated. 2 Circulating antibodies that
From the Department of Ophthalmology, Univer sity of Pittsburgh School of Medicine, and Eye and Ear Hospital, Pittsburgh, Pennsylvania. This study was supported in part by grants EY02894-02 (Dr. Mondino) and EY01991 (Dr. Brown) from the Na tional Eye Institute. Reprint requests to Bartly J. Mondino, M.D., Department of Ophthalmology, Eye and Ear Hospi tal, 230 Lothrop St., Pittsburgh, PA 15213. 536
bind to the conjunctival epithelium, but not to its basement membrane, have also been detected. 4 , 5 The association of ocular cicatricial pemphigoid with HLA-B12 an tigen suggests that there is an immunogenetic susceptibility to the development of this disease. 6 We have studied T (thymus-derived) and B (bone marrow-derived) lymphocyte enumerations in the peripheral blood of patients with ocular cicatricial pemphi goid. S U B J E C T S AND M E T H O D S
Between October 1979 and October 1980, we studied 36 patients (24 women and 12 men) with ocular cicatricial pem phigoid who were not receiving systemic corticosteroids or other immunosuppressives (Table 1). Their ages ranged from 44 to 87 years (mean age, 69.9 ± 11.2 years). We used detailed histories and ophthalmologic examinations to exclude other possible causes of conjunctival shrinkage such as radiation, severe chemical burns, acute and self-limited infections caused by adenovirus and ß-hemolytic Streptococcus, topical medications used to treat glaucoma, and Stevens-Johnson syn drome, which causes conjunctival shrink age which, unlike that of ocular cicatricial pemphigoid, is not chronically progres sive. 7
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TABLE 1 PERCENTAGES AND ABSOLUTE NUMBERS OF T AND B LYMPHOCYTES IN PATIENTS WITH OCULAR CICATRICIAL PEMPHIGOID
Patient No., Sex, Age (yrs) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
M, 54 F , 80 F, 54 F , 80 F , 67 M, 69 F , 86 M, 62 M, 86 M, 67 F , 62 F , 73 F , 60 F , 83 F , 62 M, 63 F , 80 F , 70 M, 85 F , 78 M, 87 M, 76 F , 53 F, 76 F , 66 F , 86 F , 73 M, 44 F , 54 M, 81 F , 69 M, 65 F , 64 F , 78 F , 62 F , 64
Lympho cytes 2,210 1,910 2,027 987 1,316 810 780 1,261 1,177 2,646 2,282 1,342 3,040 2,040 2,776 4,704 3,432 3,234 2,820 2,184 1,148 1,014 1,827 1,188 1,438 1,454 1,938 1,015 2,088 1,767 792 2,457 1,173 1,751 1,387 2,280
B Lymphocytes
T Lymphocytes, E Rosettes % No.
EAC Rosettes No. %
679.5 687.4 1,287.3 472.6 737.1 458.5 174.9 480.1 308.5 1,362.7 998.8 840.4 1,322.5 1,322.5 1,551.4 1,034.9 1,061.7 483.5 951.8 51.7 625.4 442.8 1,269.8 852.4 794.2 857.6 741.1 233.1 887.4 459.4 312.8 896.8 478.0 910.5 443.8 1,322.4
243.1 343.7 450.0 100.7 167.7 81.0 50.7 110.3 109.1 157.4 262.4 53.7 214.2 208.2 214.2 385.7 111.5 169.8 310.2 311.2 211.7 157.2 191.8 178.4 158.1 50.9 435.9 183.8 292.3 282.7 67.3 282.6 99.7 210.1 270.5 228.0
30.7 36.0 63.5 47.9 56.0 56.6 22.4 38.1 26.2 51.5 43.8 62.6 55.8 64.8 55.9 22.0 30.9 15.0 33.8 23.4 54.5 43.7 69.5 71.8 55.3 59.0 38.3 21.3 42.4 26.0 39.5 36.5 40.8 52.0 32.0 58.0
In addition to conjunctival shrinkage, 11 of the 36 patients (31%) showed in volvement of other mucous membranes or skin. We also studied 49 controls (27 women and 22 men). Their ages ranged from 55 to 85 years (mean age, 67.8 ± 7 . 7 years). These individuals had no external ocular diseases and no chronic systemic diseases and were not being treated with systemic corticosteroids or other immunosuppressives.
11.0 18.0 22.2 10.2 12.7 10.0 6.5 8.8 9.3 6.0 11.5 4.0 9.9 10.5 7.5 8.2 3.3 5.3 11.0 14.3 18.5 15.5 10.5 15.0 11.0 3.5 22.5 17.5 14.0 16.0 8.5 11.5 8.5 12.0 19.5 10.0
EA Rosettes No. % 66.3 104.1 176.4 66.6 55.9 8.1 30.4 37.8 214.8 79.1 22.8 57.6 30.8 41.6 40.8 163.2 171.6 48.5 112.8 152.9 114.8 30.4 100.5 83.2 71.9 130.8 58.1 21.0 83.5 88.4 31.7 49.1 24.6 87.6 52.0 114.0
3.0 5.5 8.7 6.8 4.3 1.0 3.9 3.0 18.3 3.0 1.0 4.3 1.0 2.0 1.5 3.5 5.0 1.5 4.0 7.0 10.0 3.0 5.5 7.0 5.0 9.0 3.0 2.0 4.0 5.0 4.0 2.0 2.1 5.0 3.8 5.0
Immunobeads % No. 193.4 348.5 361.4 169.1 157.6 167.3 101.4 185.4 284.8 481.8 432.1 181.6 448.1 388.7 270.3 517.4 308.9 347.7 595.0 393.1 114.8 65.9 255.8 188.3 320.7 109.0 474.7 273.0 375.8 212.0 158.4 577.4 146.6 367.7 374.5 330.6
9.5 18.3 17.8 17.1 12.0 20.7 13.0 14.7 24.2 18.2 18.9 13.5 14.7 13.3 14.0 11.0 9.0 10.8 21.1 18.0 10.0 6.5 14.0 15.9 8.0 7.5 24.5 26.0 18.0 12.0 20.0 23.5 12.5 21.0 27.0 14.5
We obtained heparinized peripheral venous blood samples from the subjects and the controls. Technicians performed the lymphocyte enumerations without knowing from which group the blood samples had come. We separated mononuclear cells by layering 3 to 5 ml of hep arinized peripheral venous blood over 3 ml of Ficoll-Hypaque solution in a plastic tube with a rounded bottom. Next, we centrifuged the tube at 2,000 rpm
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for 20 minutes at 25 C. After centrifugation, we collected the lymphocyte-rich zones, washed the lymphocytes three times in medium 199 supplemented with Hepes buffer and gentamicin, and then resuspended them in the same medium. Of the cells in these preparations, 90% to 95% were lymphocytes. We evaluated the viability of the lym phocytes by trypan blue dye exclusion, and it was higher than 95%. We adjusted the lymphocytes to three different con centrations: (1) 4 x 106 cells/ml for sheep erythrocyte rosettes (E rosettes); (2) 2 X 106 cells/ml for sheep erythrocyte coated by antibody rosettes (EA rosettes) and sheep erythrocyte coated by anti body and complement rosettes (EAC ro settes); and (3) 3 X 106 cells/ml for immunobead rosettes. We performed E rosette tests for T cell determinations by the method of Jondal, Wigzell, and Aiuti, 8 with some modifica tions. Sheep erythrocytes in Alsever's solution were washed three times in Roswell Park Memorial Institute 1640 medi um and adjusted to a 1% solution. We added 1 ml of the mixture, containing 4 x 106 lymphocytes, to a glass culture tube, and the tube was centrifuged for five minutes at 1,200 rpm. We decanted the supernatant and added 1 ml of gelatin-barbital-buffer to the cell pellet which was then gently resuspended. We mixed 0.25 ml of the cell suspension with an equal volume of 1% sheep erythrocyte indicator cells in another tube, and centrifuged the mix ture for five minutes at 500 rpm and then incubated it at 4 C for 18 to 22 hours. We then gently tapped the cells after adding two drops of 0.2% toluidine blue. We filled a hemocytometer with this mix ture and studied 200 lymphocytes, count ing the cells as positive for rosettes when three or more sheep erythrocyte indica tor cells were bound to the lymphocytes. We performed EA and EAC rosette tests for B cell determinations by the
OCTOBER, 1981
method of Ehlenberger and Nussenzweig. 9 We washed the sheep erythrocytes three times with 1640 medium and ad justed them to a 5% suspension in 1640 medium. We mixed equal volumes (1 ml) of 5% sheep erythrocytes and trypsin solution (2 mg/ml in 1640 medium) in each of two glass culture tubes. The tubes were then incubated at 37 C for one hour with occasional agitation. After the incubation, we added 0.2 ml of soybean trypsin inhibitor (10 mg/ml) to each tube. We centrifuged the tubes and washed the cell pellets four times in 1640 medium. After the fourth wash, we added 1 ml of 1640 medium to the cell pellet of each tube and resuspended the cells. We then added 1 ml of a subagglutinating dose of IgG (7S) antibodies to sheep erythrocytes to the 1 ml-cell suspension in one tube, labeled EA. We added 1 ml of a subagglutinating dose of IgM (19S) antibodies to sheep erythrocytes to the other, labeled EAC. We incubated both tubes at 37 C for 20 minutes and centri fuged them at 3,400 rpm for one minute. The cells were then gently resuspend ed in dextrose-gelatin-barbital-buffered saline with calcium and magnesium added. We then centrifuged the tubes again for one minute at 3,400 rpm. To the EA pellet, we added 10 ml of 1640 medi um. The EA indicator cells were then ready to use. To prepare EAC, we added 1 ml of the dextrose-gelatin-barbital mixture to the EA pellet in the tube labeled EAC, add ing 1 ml of diluted mouse serum (1:10 in the dextrose-gelatin-barbital mixture) as a source of complement. After the cells were incubated at 37 C for 40 minutes, we centrifuged them, washed them three times in 1640 medium, and resuspended them with 10 ml of 1640 medium. For EA and EAC rosettes, we mixed 0.3 ml of the lymphocyte suspension (2 X 106 cells/ml) with an equal volume of either EA or EAC indicator cells. The cell mixtures were then centrifuged at 1,000
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rpm for five minutes. We determined the percentages of rosette-forming cells with the procedure used in the E rosette test. We performed the immunobead ro sette test for the determination of B cells by the method of Chao and Yokoyama.10 We mixed 0.2 ml of lymphocyte suspen sion (3 x 106 cells/ml) with an equal volume of immunobeads coated with rabbit antihuman IgG, IgA, and IgM (150 X 106 beads/ml). We incubated this mixture at 37 C for 15 minutes and then centrifuged it at 800 rpm for one minute. We scored lymphocytes surrounded by three or more immunobeads as positive. The absolute numbers of T and B lym phocytes can be calculated when the white blood cell count, the percentage of lymphocytes in it, and the percentages of T and B lymphocytes are known. We evaluated differences in both percentages and absolute numbers of T and B lympho cytes between patient and control popu lations using Student's f-test. RESULTS
We found a statistically significant de crease (P<.001) in both the mean per centage and absolute n u m b e r of T lym phocytes, determined by E rosettes, in
539
patients with ocular cicatricial pemphigoid compared to age-matched controls (Table 2). We also found a statistically significant decrease in the mean percent ages (P<.05) and absolute numbers (P<.01) of B lymphocytes, determined by EAC and EA rosettes, in patients com pared to age-matched controls. Although the mean percentage and absolute num ber of B lymphocytes, determined by immunobeads, were lower in patients than in controls, the differences were not statistically significant. There was no sta tistically significant difference in mean lymphocyte counts between patients and controls. There were no statistically significant differences in mean percentages and ab solute numbers of T and B lymphocytes between patients who had only conjunctival involvement and those who had in volvement of other mucous membranes or skin in addition to conjunctival in volvement (Table 3). Table 4 shows the mean values of T and B lymphocytes in male and female patients. The only statis tically significant differences between men and women were the mean percent ages and absolute numbers of T lympho cytes, both of which were decreased in
TABLE 2 COMPARISON O F RESULTS F O R PATIENTS AND CONTROLS
Patients (No. = 36)
Controls (No. = 49)
1,880 ± 866
2,013 ± 817
> .1
%
784.9 ± 363.1 43.8 ± 15.3
1,143 ± 518.7 55.0 ± 7.5
< .001 < .001
%
204.3 ± 104.4 11.5 ± 4.9
292.9 ± 145.8 14.0 ± 5.0
< .01 < .05
%
78.4 ± 50.5 4.5 ± 3.3
130.4 ± 87.5 6.3 ± 3.9
< .01 < .05
%
296.6 ± 139.6 16.1 ± 5.3
348.4 ± 197.5 16.4 ± 6 . 1
> .1 > .5
Lymphocytes Mean count T cells E rosettes No. B cells EAC rosettes No. EA rosettes No. Immunobeads No.
P Value
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TABLE 3 COMPARISON OF RESULTS FOR PATIENTS WITH CONJUNCTIVAL INVOLVEMENT ONLY AND THOSE WITH INVOLVEMENT OF OTHER MUCOUS MEMBRANES OR SKIN AS WELL AS CONJUNCTIVA
Lymphocytes T cells E rosettes No. % B cells EAC rosettes No. % EA rosettes No. % Immunobeads No. %
Conjunctiva Only (No. = 25)
Conjunctiva Plus Other Involvement (No. = 11)
P Value
763.6 ± 381.2 43.6 ± 13.9
833.1 ± 329.9 44.3 ± 18.7
> .5 > .5
188.4 ± 114.9 11.1 ± 5.2
240.5 ± 66.3 12.9 ± 3.3
> .1 > .1
75.2 ± 54.2 4.5 ± 3.6
85.7 ± 42.5 4.6 ± 2.5
> .5 > .5
296.4 ± 135.5 16.1 ± 5.4
297.3 ± 155.4 15.0 ± 5.2
> .5 > .5
men (P<.05). There were no statistically significant differences in the mean values of T and B lymphocytes between men and women in the control group. DISCUSSION
Alterations in the proportions of T and B cells have been reported with systemic diseases. A recent study of patients with Graves' disease showed that patients with
infiltrative ophthalmopathy without pre vious antithyroid therapy and euthyroid patients with progressive ophthalmopa thy had decreased percentages of T cells compared to thyrotoxic patients without ocular disease or to controls. 11 Patients with rheumatoid arthritis demonstrated a wide range of peripheral blood T cell values with low percentages of peripheral blood T cells correlating to
TABLE 4 COMPARISON OF RESULTS FOR MEN AND WOMEN
Lymphocytes T cells E rosettes No. % B cells EAC rosettes No. % EA rosettes No. % Immunobeads No. %
Men (No. = 12)
Women (No. = 24)
P Value
626.2 ± 331.2 36.7 ± 12.5
864.2 ± 358.3 47.4 ± 15.5
< .05 < .05
194.3 ± 95.1 11.9 ± 4.0
209.3 ± 110.4 11.3 ± 5.4
80.1 ± 63.0 4.8 ± 4.8
77.6 ± 44.6 4.4 ± 2.3
> > > >
269.8 ± 170.5 16.5 ± 6.6
310.1 ± 123.3 16.0 ± 4.6
.5 .5 .5 .5
> .1 > .5
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some extent with severe clinical disease. 12 Patients with systemic lupus erythematosus showed reduced percentages and absolute numbers of peripheral blood T cells that correlated with clinical disease activity. 13 Goto and associates 14 found a slight decrease in the proportion of T cells in the peripheral blood in patients with Werner's syndrome, a condition characterized by accelerated aging; they found a similar pattern in healthy donors more than 90 years old. Wybran and Fudenberg 16 found a decrease in T cells in patients with neoplasms and with certain viral illnesses. Most patients with Sjögren's syndrome showed an increase in peripheral blood B cells; there was a decrease in T cells in some. 16 Patients with lepromatous leprosy showed re duced percentages of both T and B pe ripheral blood lymphocytes. 17 Decreased percentages and absolute numbers of T and B lymphocytes are immunologie abnormalities associated with ocular cicatricial pemphigoid. These lymphocytic abnormalities may have im portant implications in the immunopathogenesis of the disease. The reduc tion in T lymphocytes may reflect a disproportionate loss of suppressor com pared to helper T cells. Autoimmune diseases such as systemic lupus erythematosus and juvenile rheumatoid arthri tis have been associated with a loss of suppressor cells. 18,19 With reduced numbers of suppressor T cells, T and B cell responses to host tissues such as the conjunctiva may not be properly regulated or suppressed so that destructive autoimmune phenomena develop to them. It is also possible that the reduction of circulating lymphocytes in this disease results from sequestration of these cells beneath the skin and mu cous membranes. Specimens of skin and mucous membranes, including the con junctiva, show an infiltration beneath the epithelium consisting primarily of lym
PEMPHIGOID
541
phocytes and plasma cells with occasional eosinophils and a few polymorphonuclear leukocytes. 20 " 22 B lymphocytes seques tered beneath the skin and mucous mem branes may be converted to plasma cells, producing antibodies against these tis sues. REFERENCES 1. Rook, A., Wilkinson, D. S., and Ebling, F. J. G. : Textbook of Dermatology. Oxford, Blackwell Scientific Publications, 1968, vol. 2, pp. 11631192. 2. Griffith, M. R., Fukuyama, K., Tuffanelli, D., and Silverman, S.: Immunofluorescent studies in mucous membrane pemphigoid. Arch. Dermatol. 109:195, 1974. 3. Rogers, R. S., Perry, H. O., Bean, S. F., and Jordan, R. E.: Immunopathology of cicatricial pem phigoid. Studies of complement deposition. J. In vest. Dermatol. 68:39, 1977. 4. Mondino, B. J., Ross, A. N., Rabin, B. S., and Brown, S. I.: Autoimmune phenomena in ocular cicatricial pemphigoid. Am. J. Ophthalmol. 83:443, 1977. 5. Mondino, B. J., Brown, S. I., and Rabin, B. S.: Autoimmune phenomena of the external eye. Oph thalmology 85:801, 1978. 6. : HLA antigens in ocular cicatricial pem phigoid. Arch. Ophthalmol. 97:470, 1979. 7. Mondino, B. J. : Bullous diseases of the skin and mucous membranes. In Duane, T. (ed.): Clinical Ophthalmology. Hagerstown, Harper and Row, 1980, vol. 4, pp. 1-16. 8. Jondal, M., Wigzell, H., and Aiuti, F.: Human lymphocyte subpopulations. Classification according to surface markers and/or functional characteristics. Transplant Rev. 16:163, 1973. 9. Ehlenberger, A. G., and Nussenzweig, V. : Identification ofcells with complement receptors. In Bloom, B. R., and David, J. R. (eds.): In Vitro Method of Cell-Mediated and Tumor Immunity. New York, Academic Press, 1976, pp. 113-121. 10. Chao, W., andYokoyama, M. M.: Determina tion of B lymphocyte population using antibodycoated polyacrylamide beads. Clin. Chim. Acta 78:79, 1977. 11. Sergott, R. C , Felberg, N. T., Savirio, P. J., Blizzard, J. J., and Schatz, N. J.: E-rosette formation in Graves' ophthalmopathy. Invest. Ophthalmol. Vis. Sei. 18:1245, 1979. 12. Williams, R. C , DeBoard, J. R., Mellbye, O. J., Messner, R. P., and Lindström, F. D.: Stud ies of T- and B-lymphocytes in patients with connec tive tissue diseases. J. Clin. Invest. 52:283, 1973. 13. Messner, R. P., Lindström, F. D., and Wil liams, R. C : Peripheral blood lymphocyte cell sur face markers during the course of systemic lupus erythematosus. J. Clin. Invest. 52:3046, 1973. 14. Goto, M., Horiuchi, Y., Okumura, K., Tada,
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T., and Ohmori, K. : Immunological abnormalities of aging. An analysis of T lymphocyte subpopulations of Werner's syndrome. J. Clin. Invest. 64:695, 1979. 15. Wybran, J., and Fudenberg, H. H.: Thymusderived rosette-forming cells in various human dis ease states. Cancer, lymphoma, bacterial and viral infections, and other diseases. J. Clin. Invest. 52:1026, 1973. 16. Talal, N., Sylvester, R. A., Daniels, T. E., Greenspan, J. S., and Williams, R. C : T and B lymphocytes in peripheral blood and tissue lesions in Sjögren's syndrome. J. Clin. Invest. 53:180, 1974. 17. Mendes, N. F., Kopersztych, S., and Mota, N. G. S.: T and B lymphocytes in patients with lepromatous leprosy. Clin. Exp. Immunol. 16:23, 1974. 18. Goodwin, J. S., and Williams, R. C : Sup
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pressor cells—A recent conceptual epidemic. J. Clin. Lab. Immunol. 2:89, 1979. 19. Strelkauskas, A. J., Callery, R. T., McDow ell, J., Borel, Y., and Schlossman, S. F.: Direct evidence for loss of human suppressor cells during active autoimmune disease. Proc. Natl. Acad. Sei. USA 75:5150, 1978. 20. Norn, M. S., and Kristensen, E. B.: Benign mucous membrane pemphigoid. II. Cytology. Acta Ophthalmol. 52:282, 1974. 21. Andersen, S. R., Jensen, O. A., Kristensen, E. B., and Norn, M. S.: Benign mucous membrane pemphigoid. III. Biopsy. Acta Ophthalmol. 52:455, 1974. 22. Rook, A., Wilkinson, D. S., and Ebling, F. J. G. : Textbook of Dermatology. Oxford, Blackwell Scientific Publications, 1968, vol. 2, p. 1182.
O P H T H A L M I C MINIATURE
"Well, it's a nasty thing, Ted. Entropion is where the eyelids are turned in and the lashes rub against the eyeball. Causes a lot of pain, even ulcération and blindness. Even a mild case is damned uncomfortable for a dog." "Poor awd bugger. Could an operation cure it?" "Yes, Ted, it's one of the most satisfying jobs a vet can d o . " James Herriot, All Things Wise and Wonderful
H I A H W A R A O , M.S.,
A N D S T U A R T I. B R O W N ,
M.D.
Pittsburgh, Pennsylvania
We performed peripheral blood T and B lymphocyte enumerations on 36 patients with ocular cicatricial pemphigoid and 49 age-matched controls. We found a decrease in both the mean percentage and the absolute number of T lymphocytes (determined by E rosettes) in patients compared to controls. We also found a decrease in the mean percentages and absolute numbers of B lymphocytes (determined by EAC and EA rosettes) in patients compared to controls.
Cicatricial pemphigoid is a chronic dis ease characterized by recurrent blisters or bullae of the skin and mucous mem branes and a tendency for scar forma tion. 1 Characteristic features of ocular involvement include progressive shrink age of the conjunctiva, entropion, trichiasis, xerosis, and, ultimately, reduced vision from corneal opacification. Im munologie abnormalities are associated with this disease. In cicatricial pemphi goid, immunoglobulins and components of both the alternative and classical com plement pathways are bound to the base ment membrane zone of skin and oral mucosa. 2,3 Immunopathologic studies of the conjunctiva showed immunoglobulins and the third component of complement bound not only to the conjunctival base ment membrane but also to its epitheli um. 4 ' 5 Circulating antibodies that bind to the basement membrane zone of skin and oral mucosa have occasionally been dem onstrated. 2 Circulating antibodies that
From the Department of Ophthalmology, Univer sity of Pittsburgh School of Medicine, and Eye and Ear Hospital, Pittsburgh, Pennsylvania. This study was supported in part by grants EY02894-02 (Dr. Mondino) and EY01991 (Dr. Brown) from the Na tional Eye Institute. Reprint requests to Bartly J. Mondino, M.D., Department of Ophthalmology, Eye and Ear Hospi tal, 230 Lothrop St., Pittsburgh, PA 15213. 536
bind to the conjunctival epithelium, but not to its basement membrane, have also been detected. 4 , 5 The association of ocular cicatricial pemphigoid with HLA-B12 an tigen suggests that there is an immunogenetic susceptibility to the development of this disease. 6 We have studied T (thymus-derived) and B (bone marrow-derived) lymphocyte enumerations in the peripheral blood of patients with ocular cicatricial pemphi goid. S U B J E C T S AND M E T H O D S
Between October 1979 and October 1980, we studied 36 patients (24 women and 12 men) with ocular cicatricial pem phigoid who were not receiving systemic corticosteroids or other immunosuppressives (Table 1). Their ages ranged from 44 to 87 years (mean age, 69.9 ± 11.2 years). We used detailed histories and ophthalmologic examinations to exclude other possible causes of conjunctival shrinkage such as radiation, severe chemical burns, acute and self-limited infections caused by adenovirus and ß-hemolytic Streptococcus, topical medications used to treat glaucoma, and Stevens-Johnson syn drome, which causes conjunctival shrink age which, unlike that of ocular cicatricial pemphigoid, is not chronically progres sive. 7
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TABLE 1 PERCENTAGES AND ABSOLUTE NUMBERS OF T AND B LYMPHOCYTES IN PATIENTS WITH OCULAR CICATRICIAL PEMPHIGOID
Patient No., Sex, Age (yrs) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
M, 54 F , 80 F, 54 F , 80 F , 67 M, 69 F , 86 M, 62 M, 86 M, 67 F , 62 F , 73 F , 60 F , 83 F , 62 M, 63 F , 80 F , 70 M, 85 F , 78 M, 87 M, 76 F , 53 F, 76 F , 66 F , 86 F , 73 M, 44 F , 54 M, 81 F , 69 M, 65 F , 64 F , 78 F , 62 F , 64
Lympho cytes 2,210 1,910 2,027 987 1,316 810 780 1,261 1,177 2,646 2,282 1,342 3,040 2,040 2,776 4,704 3,432 3,234 2,820 2,184 1,148 1,014 1,827 1,188 1,438 1,454 1,938 1,015 2,088 1,767 792 2,457 1,173 1,751 1,387 2,280
B Lymphocytes
T Lymphocytes, E Rosettes % No.
EAC Rosettes No. %
679.5 687.4 1,287.3 472.6 737.1 458.5 174.9 480.1 308.5 1,362.7 998.8 840.4 1,322.5 1,322.5 1,551.4 1,034.9 1,061.7 483.5 951.8 51.7 625.4 442.8 1,269.8 852.4 794.2 857.6 741.1 233.1 887.4 459.4 312.8 896.8 478.0 910.5 443.8 1,322.4
243.1 343.7 450.0 100.7 167.7 81.0 50.7 110.3 109.1 157.4 262.4 53.7 214.2 208.2 214.2 385.7 111.5 169.8 310.2 311.2 211.7 157.2 191.8 178.4 158.1 50.9 435.9 183.8 292.3 282.7 67.3 282.6 99.7 210.1 270.5 228.0
30.7 36.0 63.5 47.9 56.0 56.6 22.4 38.1 26.2 51.5 43.8 62.6 55.8 64.8 55.9 22.0 30.9 15.0 33.8 23.4 54.5 43.7 69.5 71.8 55.3 59.0 38.3 21.3 42.4 26.0 39.5 36.5 40.8 52.0 32.0 58.0
In addition to conjunctival shrinkage, 11 of the 36 patients (31%) showed in volvement of other mucous membranes or skin. We also studied 49 controls (27 women and 22 men). Their ages ranged from 55 to 85 years (mean age, 67.8 ± 7 . 7 years). These individuals had no external ocular diseases and no chronic systemic diseases and were not being treated with systemic corticosteroids or other immunosuppressives.
11.0 18.0 22.2 10.2 12.7 10.0 6.5 8.8 9.3 6.0 11.5 4.0 9.9 10.5 7.5 8.2 3.3 5.3 11.0 14.3 18.5 15.5 10.5 15.0 11.0 3.5 22.5 17.5 14.0 16.0 8.5 11.5 8.5 12.0 19.5 10.0
EA Rosettes No. % 66.3 104.1 176.4 66.6 55.9 8.1 30.4 37.8 214.8 79.1 22.8 57.6 30.8 41.6 40.8 163.2 171.6 48.5 112.8 152.9 114.8 30.4 100.5 83.2 71.9 130.8 58.1 21.0 83.5 88.4 31.7 49.1 24.6 87.6 52.0 114.0
3.0 5.5 8.7 6.8 4.3 1.0 3.9 3.0 18.3 3.0 1.0 4.3 1.0 2.0 1.5 3.5 5.0 1.5 4.0 7.0 10.0 3.0 5.5 7.0 5.0 9.0 3.0 2.0 4.0 5.0 4.0 2.0 2.1 5.0 3.8 5.0
Immunobeads % No. 193.4 348.5 361.4 169.1 157.6 167.3 101.4 185.4 284.8 481.8 432.1 181.6 448.1 388.7 270.3 517.4 308.9 347.7 595.0 393.1 114.8 65.9 255.8 188.3 320.7 109.0 474.7 273.0 375.8 212.0 158.4 577.4 146.6 367.7 374.5 330.6
9.5 18.3 17.8 17.1 12.0 20.7 13.0 14.7 24.2 18.2 18.9 13.5 14.7 13.3 14.0 11.0 9.0 10.8 21.1 18.0 10.0 6.5 14.0 15.9 8.0 7.5 24.5 26.0 18.0 12.0 20.0 23.5 12.5 21.0 27.0 14.5
We obtained heparinized peripheral venous blood samples from the subjects and the controls. Technicians performed the lymphocyte enumerations without knowing from which group the blood samples had come. We separated mononuclear cells by layering 3 to 5 ml of hep arinized peripheral venous blood over 3 ml of Ficoll-Hypaque solution in a plastic tube with a rounded bottom. Next, we centrifuged the tube at 2,000 rpm
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for 20 minutes at 25 C. After centrifugation, we collected the lymphocyte-rich zones, washed the lymphocytes three times in medium 199 supplemented with Hepes buffer and gentamicin, and then resuspended them in the same medium. Of the cells in these preparations, 90% to 95% were lymphocytes. We evaluated the viability of the lym phocytes by trypan blue dye exclusion, and it was higher than 95%. We adjusted the lymphocytes to three different con centrations: (1) 4 x 106 cells/ml for sheep erythrocyte rosettes (E rosettes); (2) 2 X 106 cells/ml for sheep erythrocyte coated by antibody rosettes (EA rosettes) and sheep erythrocyte coated by anti body and complement rosettes (EAC ro settes); and (3) 3 X 106 cells/ml for immunobead rosettes. We performed E rosette tests for T cell determinations by the method of Jondal, Wigzell, and Aiuti, 8 with some modifica tions. Sheep erythrocytes in Alsever's solution were washed three times in Roswell Park Memorial Institute 1640 medi um and adjusted to a 1% solution. We added 1 ml of the mixture, containing 4 x 106 lymphocytes, to a glass culture tube, and the tube was centrifuged for five minutes at 1,200 rpm. We decanted the supernatant and added 1 ml of gelatin-barbital-buffer to the cell pellet which was then gently resuspended. We mixed 0.25 ml of the cell suspension with an equal volume of 1% sheep erythrocyte indicator cells in another tube, and centrifuged the mix ture for five minutes at 500 rpm and then incubated it at 4 C for 18 to 22 hours. We then gently tapped the cells after adding two drops of 0.2% toluidine blue. We filled a hemocytometer with this mix ture and studied 200 lymphocytes, count ing the cells as positive for rosettes when three or more sheep erythrocyte indica tor cells were bound to the lymphocytes. We performed EA and EAC rosette tests for B cell determinations by the
OCTOBER, 1981
method of Ehlenberger and Nussenzweig. 9 We washed the sheep erythrocytes three times with 1640 medium and ad justed them to a 5% suspension in 1640 medium. We mixed equal volumes (1 ml) of 5% sheep erythrocytes and trypsin solution (2 mg/ml in 1640 medium) in each of two glass culture tubes. The tubes were then incubated at 37 C for one hour with occasional agitation. After the incubation, we added 0.2 ml of soybean trypsin inhibitor (10 mg/ml) to each tube. We centrifuged the tubes and washed the cell pellets four times in 1640 medium. After the fourth wash, we added 1 ml of 1640 medium to the cell pellet of each tube and resuspended the cells. We then added 1 ml of a subagglutinating dose of IgG (7S) antibodies to sheep erythrocytes to the 1 ml-cell suspension in one tube, labeled EA. We added 1 ml of a subagglutinating dose of IgM (19S) antibodies to sheep erythrocytes to the other, labeled EAC. We incubated both tubes at 37 C for 20 minutes and centri fuged them at 3,400 rpm for one minute. The cells were then gently resuspend ed in dextrose-gelatin-barbital-buffered saline with calcium and magnesium added. We then centrifuged the tubes again for one minute at 3,400 rpm. To the EA pellet, we added 10 ml of 1640 medi um. The EA indicator cells were then ready to use. To prepare EAC, we added 1 ml of the dextrose-gelatin-barbital mixture to the EA pellet in the tube labeled EAC, add ing 1 ml of diluted mouse serum (1:10 in the dextrose-gelatin-barbital mixture) as a source of complement. After the cells were incubated at 37 C for 40 minutes, we centrifuged them, washed them three times in 1640 medium, and resuspended them with 10 ml of 1640 medium. For EA and EAC rosettes, we mixed 0.3 ml of the lymphocyte suspension (2 X 106 cells/ml) with an equal volume of either EA or EAC indicator cells. The cell mixtures were then centrifuged at 1,000
OCULAR CICATRICIAL PEMPHIGOID
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rpm for five minutes. We determined the percentages of rosette-forming cells with the procedure used in the E rosette test. We performed the immunobead ro sette test for the determination of B cells by the method of Chao and Yokoyama.10 We mixed 0.2 ml of lymphocyte suspen sion (3 x 106 cells/ml) with an equal volume of immunobeads coated with rabbit antihuman IgG, IgA, and IgM (150 X 106 beads/ml). We incubated this mixture at 37 C for 15 minutes and then centrifuged it at 800 rpm for one minute. We scored lymphocytes surrounded by three or more immunobeads as positive. The absolute numbers of T and B lym phocytes can be calculated when the white blood cell count, the percentage of lymphocytes in it, and the percentages of T and B lymphocytes are known. We evaluated differences in both percentages and absolute numbers of T and B lympho cytes between patient and control popu lations using Student's f-test. RESULTS
We found a statistically significant de crease (P<.001) in both the mean per centage and absolute n u m b e r of T lym phocytes, determined by E rosettes, in
539
patients with ocular cicatricial pemphigoid compared to age-matched controls (Table 2). We also found a statistically significant decrease in the mean percent ages (P<.05) and absolute numbers (P<.01) of B lymphocytes, determined by EAC and EA rosettes, in patients com pared to age-matched controls. Although the mean percentage and absolute num ber of B lymphocytes, determined by immunobeads, were lower in patients than in controls, the differences were not statistically significant. There was no sta tistically significant difference in mean lymphocyte counts between patients and controls. There were no statistically significant differences in mean percentages and ab solute numbers of T and B lymphocytes between patients who had only conjunctival involvement and those who had in volvement of other mucous membranes or skin in addition to conjunctival in volvement (Table 3). Table 4 shows the mean values of T and B lymphocytes in male and female patients. The only statis tically significant differences between men and women were the mean percent ages and absolute numbers of T lympho cytes, both of which were decreased in
TABLE 2 COMPARISON O F RESULTS F O R PATIENTS AND CONTROLS
Patients (No. = 36)
Controls (No. = 49)
1,880 ± 866
2,013 ± 817
> .1
%
784.9 ± 363.1 43.8 ± 15.3
1,143 ± 518.7 55.0 ± 7.5
< .001 < .001
%
204.3 ± 104.4 11.5 ± 4.9
292.9 ± 145.8 14.0 ± 5.0
< .01 < .05
%
78.4 ± 50.5 4.5 ± 3.3
130.4 ± 87.5 6.3 ± 3.9
< .01 < .05
%
296.6 ± 139.6 16.1 ± 5.3
348.4 ± 197.5 16.4 ± 6 . 1
> .1 > .5
Lymphocytes Mean count T cells E rosettes No. B cells EAC rosettes No. EA rosettes No. Immunobeads No.
P Value
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TABLE 3 COMPARISON OF RESULTS FOR PATIENTS WITH CONJUNCTIVAL INVOLVEMENT ONLY AND THOSE WITH INVOLVEMENT OF OTHER MUCOUS MEMBRANES OR SKIN AS WELL AS CONJUNCTIVA
Lymphocytes T cells E rosettes No. % B cells EAC rosettes No. % EA rosettes No. % Immunobeads No. %
Conjunctiva Only (No. = 25)
Conjunctiva Plus Other Involvement (No. = 11)
P Value
763.6 ± 381.2 43.6 ± 13.9
833.1 ± 329.9 44.3 ± 18.7
> .5 > .5
188.4 ± 114.9 11.1 ± 5.2
240.5 ± 66.3 12.9 ± 3.3
> .1 > .1
75.2 ± 54.2 4.5 ± 3.6
85.7 ± 42.5 4.6 ± 2.5
> .5 > .5
296.4 ± 135.5 16.1 ± 5.4
297.3 ± 155.4 15.0 ± 5.2
> .5 > .5
men (P<.05). There were no statistically significant differences in the mean values of T and B lymphocytes between men and women in the control group. DISCUSSION
Alterations in the proportions of T and B cells have been reported with systemic diseases. A recent study of patients with Graves' disease showed that patients with
infiltrative ophthalmopathy without pre vious antithyroid therapy and euthyroid patients with progressive ophthalmopa thy had decreased percentages of T cells compared to thyrotoxic patients without ocular disease or to controls. 11 Patients with rheumatoid arthritis demonstrated a wide range of peripheral blood T cell values with low percentages of peripheral blood T cells correlating to
TABLE 4 COMPARISON OF RESULTS FOR MEN AND WOMEN
Lymphocytes T cells E rosettes No. % B cells EAC rosettes No. % EA rosettes No. % Immunobeads No. %
Men (No. = 12)
Women (No. = 24)
P Value
626.2 ± 331.2 36.7 ± 12.5
864.2 ± 358.3 47.4 ± 15.5
< .05 < .05
194.3 ± 95.1 11.9 ± 4.0
209.3 ± 110.4 11.3 ± 5.4
80.1 ± 63.0 4.8 ± 4.8
77.6 ± 44.6 4.4 ± 2.3
> > > >
269.8 ± 170.5 16.5 ± 6.6
310.1 ± 123.3 16.0 ± 4.6
.5 .5 .5 .5
> .1 > .5
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OCULAR CICATRICIAL
some extent with severe clinical disease. 12 Patients with systemic lupus erythematosus showed reduced percentages and absolute numbers of peripheral blood T cells that correlated with clinical disease activity. 13 Goto and associates 14 found a slight decrease in the proportion of T cells in the peripheral blood in patients with Werner's syndrome, a condition characterized by accelerated aging; they found a similar pattern in healthy donors more than 90 years old. Wybran and Fudenberg 16 found a decrease in T cells in patients with neoplasms and with certain viral illnesses. Most patients with Sjögren's syndrome showed an increase in peripheral blood B cells; there was a decrease in T cells in some. 16 Patients with lepromatous leprosy showed re duced percentages of both T and B pe ripheral blood lymphocytes. 17 Decreased percentages and absolute numbers of T and B lymphocytes are immunologie abnormalities associated with ocular cicatricial pemphigoid. These lymphocytic abnormalities may have im portant implications in the immunopathogenesis of the disease. The reduc tion in T lymphocytes may reflect a disproportionate loss of suppressor com pared to helper T cells. Autoimmune diseases such as systemic lupus erythematosus and juvenile rheumatoid arthri tis have been associated with a loss of suppressor cells. 18,19 With reduced numbers of suppressor T cells, T and B cell responses to host tissues such as the conjunctiva may not be properly regulated or suppressed so that destructive autoimmune phenomena develop to them. It is also possible that the reduction of circulating lymphocytes in this disease results from sequestration of these cells beneath the skin and mu cous membranes. Specimens of skin and mucous membranes, including the con junctiva, show an infiltration beneath the epithelium consisting primarily of lym
PEMPHIGOID
541
phocytes and plasma cells with occasional eosinophils and a few polymorphonuclear leukocytes. 20 " 22 B lymphocytes seques tered beneath the skin and mucous mem branes may be converted to plasma cells, producing antibodies against these tis sues. REFERENCES 1. Rook, A., Wilkinson, D. S., and Ebling, F. J. G. : Textbook of Dermatology. Oxford, Blackwell Scientific Publications, 1968, vol. 2, pp. 11631192. 2. Griffith, M. R., Fukuyama, K., Tuffanelli, D., and Silverman, S.: Immunofluorescent studies in mucous membrane pemphigoid. Arch. Dermatol. 109:195, 1974. 3. Rogers, R. S., Perry, H. O., Bean, S. F., and Jordan, R. E.: Immunopathology of cicatricial pem phigoid. Studies of complement deposition. J. In vest. Dermatol. 68:39, 1977. 4. Mondino, B. J., Ross, A. N., Rabin, B. S., and Brown, S. I.: Autoimmune phenomena in ocular cicatricial pemphigoid. Am. J. Ophthalmol. 83:443, 1977. 5. Mondino, B. J., Brown, S. I., and Rabin, B. S.: Autoimmune phenomena of the external eye. Oph thalmology 85:801, 1978. 6. : HLA antigens in ocular cicatricial pem phigoid. Arch. Ophthalmol. 97:470, 1979. 7. Mondino, B. J. : Bullous diseases of the skin and mucous membranes. In Duane, T. (ed.): Clinical Ophthalmology. Hagerstown, Harper and Row, 1980, vol. 4, pp. 1-16. 8. Jondal, M., Wigzell, H., and Aiuti, F.: Human lymphocyte subpopulations. Classification according to surface markers and/or functional characteristics. Transplant Rev. 16:163, 1973. 9. Ehlenberger, A. G., and Nussenzweig, V. : Identification ofcells with complement receptors. In Bloom, B. R., and David, J. R. (eds.): In Vitro Method of Cell-Mediated and Tumor Immunity. New York, Academic Press, 1976, pp. 113-121. 10. Chao, W., andYokoyama, M. M.: Determina tion of B lymphocyte population using antibodycoated polyacrylamide beads. Clin. Chim. Acta 78:79, 1977. 11. Sergott, R. C , Felberg, N. T., Savirio, P. J., Blizzard, J. J., and Schatz, N. J.: E-rosette formation in Graves' ophthalmopathy. Invest. Ophthalmol. Vis. Sei. 18:1245, 1979. 12. Williams, R. C , DeBoard, J. R., Mellbye, O. J., Messner, R. P., and Lindström, F. D.: Stud ies of T- and B-lymphocytes in patients with connec tive tissue diseases. J. Clin. Invest. 52:283, 1973. 13. Messner, R. P., Lindström, F. D., and Wil liams, R. C : Peripheral blood lymphocyte cell sur face markers during the course of systemic lupus erythematosus. J. Clin. Invest. 52:3046, 1973. 14. Goto, M., Horiuchi, Y., Okumura, K., Tada,
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T., and Ohmori, K. : Immunological abnormalities of aging. An analysis of T lymphocyte subpopulations of Werner's syndrome. J. Clin. Invest. 64:695, 1979. 15. Wybran, J., and Fudenberg, H. H.: Thymusderived rosette-forming cells in various human dis ease states. Cancer, lymphoma, bacterial and viral infections, and other diseases. J. Clin. Invest. 52:1026, 1973. 16. Talal, N., Sylvester, R. A., Daniels, T. E., Greenspan, J. S., and Williams, R. C : T and B lymphocytes in peripheral blood and tissue lesions in Sjögren's syndrome. J. Clin. Invest. 53:180, 1974. 17. Mendes, N. F., Kopersztych, S., and Mota, N. G. S.: T and B lymphocytes in patients with lepromatous leprosy. Clin. Exp. Immunol. 16:23, 1974. 18. Goodwin, J. S., and Williams, R. C : Sup
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pressor cells—A recent conceptual epidemic. J. Clin. Lab. Immunol. 2:89, 1979. 19. Strelkauskas, A. J., Callery, R. T., McDow ell, J., Borel, Y., and Schlossman, S. F.: Direct evidence for loss of human suppressor cells during active autoimmune disease. Proc. Natl. Acad. Sei. USA 75:5150, 1978. 20. Norn, M. S., and Kristensen, E. B.: Benign mucous membrane pemphigoid. II. Cytology. Acta Ophthalmol. 52:282, 1974. 21. Andersen, S. R., Jensen, O. A., Kristensen, E. B., and Norn, M. S.: Benign mucous membrane pemphigoid. III. Biopsy. Acta Ophthalmol. 52:455, 1974. 22. Rook, A., Wilkinson, D. S., and Ebling, F. J. G. : Textbook of Dermatology. Oxford, Blackwell Scientific Publications, 1968, vol. 2, p. 1182.
O P H T H A L M I C MINIATURE
"Well, it's a nasty thing, Ted. Entropion is where the eyelids are turned in and the lashes rub against the eyeball. Causes a lot of pain, even ulcération and blindness. Even a mild case is damned uncomfortable for a dog." "Poor awd bugger. Could an operation cure it?" "Yes, Ted, it's one of the most satisfying jobs a vet can d o . " James Herriot, All Things Wise and Wonderful