Epstein-Barr Virus infection and Immunologic Dysfunction in Patients with Aqueous Tear Deficiency

Epstein-Barr Virus Infection and Immunologic Dysfunction in Patients with Aqueous Tear Deficiency STEPHEN C. PFLUGFELDER, MD, 1 SCHEFFER C. G. TSENG, MD, PhD, 1 JAYS. PEPOSE, MD, PhD/ MARY ANN FLETCHER, PhD,3 NANCY KLIMAS, MD, 3 WILLIAM FEUER, MS 1•4

Abstract: The authors tested their hypothesis that Epstein-Barr virus (EBV) infection is a risk factor for aqueous tear deficiency (ATD) by evaluating 3S ATD patients and 17 controls for serologic evidence of EBV infection. Aqueous tear deficiency was graded clinically as mild or severe. A linear trend toward elevated EBV capsid (P < 0.05) and early antigen (P < 0.001) titers was noted from control to severe ATD patients. Rubella and cytomegalovirus antibody titers were poorly correlated with EBV titers, suggesting that the elevated EBV antibodies in ATD patients were not due to nonspecific polyclonal B-cell activation. Epstein-Barr virus antigens were detected in two of six lacrimal gland biopsies from severe ATD patients with Sjogren's syndrome, but in none of the control glands. Aqueous tear deficiency patients were evaluated for immunologic dysfunction associated with EBV infection. Linear trends of elevated serum lgG (P < 0.05), autoantibody and immune complex positivity (P < 0.05), and reduced natural killer cell cytotoxicity (P < 0.05) were found from controls to severe ATD patients. Furthermore, reduced T-helper lymphocyte counts (P < 0.06) and an increased percentage of HLA-DR+ CDS lymphocytes (P < 0.05) were observed in severe ATD patients compared with the mild and control groups. A multivariate analysis of the data showed a significant correlation between severe ATD and elevated EBV early antigen titers, Sjogren's syndrome, and an increased percentage of HLA-DR+ CDS lymphocytes. The authors' findings suggest that EBV infection may be a risk factor for development of ATD in a subset of A TO patients with greater disease severity, Sjogren's syndrome, and immunologic dysfunction. Ophthalmology 1990; 97:313-323

Originally received: May 3, 1989. Revision accepted: September 25, 1989. 1

Department of Ophthalmology, University of Miami School of Medicine, Miami. Departments of Ophthalmology and Pathology, Washington University, StLouis. 3 Department of Medicine, University of Miami School of Medicine, Miami. 4 Department of Biostatistics, University of Miami School of Medicine, Miami. 2

Presented in part at the Association for Research and Vision in Ophthalmology Meeting, Sarasota, May 1988. Supported in part by BSRG H578450 from the NIH (SCP), United States Public Health Service research core grant P30-E1-02180, and an unrestricted grant from Research to Prevent Blindness (JSP). Reprint requests to Stephen C. Pflugfelder, MD, Bascom Palmer Eye Institute, PO Box 016880, Miami, FL 33101.

Aqueous tear deficiency (A TO) is often associated with lymphocytic infiltration and eventual destruction of the lacrimal gland acini and ducts. This appears to be the exclusive cause of decreased tear production in patients with evidence of immune dysfunction. 1 The inciting factor for this autoimmune destruction has not been definitely established; however, there is increasing evidence that it may be in response to persistent or reactivated infection by a ubiquitous herpes group virus, Epstein-Barr virus (EBV). W e2 and others, 3 have previously reported cases of primary Sjogren's syndrome which developed immediately after infectious mononucleosis. 2 Additional evidence supports EBV as an etiologic factor inciting lymphocytic proliferation and destruction of the lacrimal gland. Epi313

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Table 1. Aqueous Tear Deficiency Severity Grading System Severity Grade Ill Fluorescein clearance test Rose Bengal staining Impression cytology Corneal complications

+

+

N

EZ

c

+

cc

EZ +NEZ or EZ + FK

IV +

cc

EZ + NEZ and CU

C = conjunctival staining; CC = conjunctival plus corneal staining; N normal; EZ = squamous metaplasia in the exposure zone; NEZ squamous metaplasia in the noriexposure zone; FK = filamentary keratitis; CU = corneal ulceration. = =

myoepithelial islands which are occasionally found in lacrimal gland biopsies from patients with aqueous tear deficiency4 have similar histologic characteristics to certain undifferentiated nasopharyngeal and thymic carcinomas found to contain EBV genomes. 5•6 B-cell lymphoproliferative disorders occur in primary Sjogren's syndrome patients at a 40-fold greater rate than the general population.7·8 Both EBV9 and human herpes type 6 (HHV-6, HBLV) 10 viral DNA have been found in these lymphomas. Epstein-Barr virus is a potent activator of proliferation and immunoglobulin production by B-cells. 11 It induces hyperglobulinemia and autoantibody production in patients with infectious mononucleosis, and it may be responsible for similar findings in ATD patients. 1·12 Significant reduction in percentage ofT-lymphocytes and natural killer cell function, both of which are involved in suppression of EBV infection, 13 have been observed in Sjogren's syndrome patients. 14- 16 Finally, Fox and associates9 have recently demonstrated EBV antigens and DNA in salivary gland biopsies of primary Sjogren syndrome patients, but not in controls. Based on our clinical observations, and the reports implicating EBV in the pathogenesis of Sjogren's syndrome, we hypothesize that EBV infection is associated with development of ATD. To test this hypothesis, we evaluated ATD patients and controls for serologic evidence of EBV infection. Lacrimal glands from selected ATD patients and controls were examined for the presence ofEBV antigens. Furthermore, ATD patients and controls were also evaluated for immune system dysfunction associated with EBV infection.

PATIENTS AND METHODS PATIENT SELECTION

All patients referred to two members (SCP, SCGT) of the Cornea-External Disease service of the University of Miami Department of Ophthalmology from February to August 1987 with symptoms of ocular irritation were evaluated for ATD by a 30-minute fluorescein clearance test. This test consists of a repetitive Schirmer test com314

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bined with a dye dilution test. Thirty-eight ATD patients were identified during this period, and all of these consented to participate in this study. A complete history and ophthalmologic examination including rose Bengal surface staining and impression cytology were performed. The latter was evaluated in a masked fashion according to previously reported criteria. 17 An ATD severity grading system (Table 1) was devised (grades I and II, mild ATD; grades III and IV, severe ATD). Xerostomia was diagnosed in patients with a history of difficulty chewing or swallowing dry foods such as crackers, and the presence of parched oral mucous membranes and decreased saliva pool on the floor of the mouth. Diagnosis of primary Sjogren's syndrome was based on criteria proposed by Fox and associates, 18 and include the following: ( 1) decreased aqueous tear production by fluorescein clearance test, (2) rose Bengal staining of the conjunctiva, or conjunctiva and cornea in the interpalpebral zone, (3) xerostomia, (4) elevated autoantibody titers (antinuclear antibodies and/or rheumatoid factor titer 2: 1: 160), and (5) absence of a connective tissue disorder. Aqueous tear deficiency patients with a connective tissue disorder, xerostomia, and elevated autoantibody titers were classified as secondary Sjogren's syndrome. Seventeen patients of similar age and sex distribution with symptoms of ocular irritation, but no ATD by fluorescein clearance testing were recruited as controls. Phlebotomy was performed on all ATD patients and controls. Blood was drawn into appropriate tubes for serology and whole blood mononuclear cell immunotyping. The laboratory analysis of these specimens was performed in a masked fashion. VIRAL SEROLOGY

Serum IgG antibodies to EBV viral capsid, and the diffuse and restricted components of early antigen were determined by indirect immunofluorescence as previously described. 19 IgM antibody to viral capsid were not routinely performed. Anti-EBV nuclear antigen titers were determined using an anti-complement immunofluorescence technique. 19 Enzyme-linked immunosorbent assays were used for detection of lgG antibodies to early and late cytomegalovirus (CMV) nuclear antigens (M.A. Rioproducts, Walkersville, MD), rubella virus (M.A. Rioproducts, Walkersville, MD), and human immunodeficiency virus type 1 (HIV, Abbott, Chicago, IL). Cytomegalovirus and rubella antibodies were assayed to determine if antibody titers to other viruses were elevated as a result of polyclonal B-cell activation. Cytomegalovirus serology also was performed to determine if there was serologic evidence of infection with another herpes group virus associated with reactivation in patients with depressed host immune defenses. Human immunodeficiency virus serology was performed in all patients with T-cell helper/suppressor ratios less than 1. HUMORAL IMMUNE EVALUATION

Assays for serum autoantibodies, hypergammaglobulinemia, and circulating immune complexes were per-

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formed since they were necessary diagnostic criteria for Sjogren's syndrome, and they have been detected in patients with EBV infections. 11 •20 Antinuclear antibodies and anti-nONA were identified by indirect immunofluorescence using Hep2 cells as substrate for antinuclear antibodies, and Crithidiae luciliae for nONA (Kallestadt Laboratories, Austin, TX). Sjogren's syndrome antibodies A and B were assayed by counterimmunoelectrophoresis. Sjogren's syndrome antibody A antigen was obtained from human spleen and Sjogren's syndrome antibody B antigen was obtained from rabbit thymus (Pel-Freeze, Rogers, AR). Rheumatoid factor was detected using the Beckman Immunochemistry System Analyzer II (Beckman Instruments, Brea, CA). Positive and negative control sera from the Centers for Disease Control (Atlanta, GA) were used for each assay. Levels of immunoglobulins lgM, lgG, and IgA and complement components (C3, C4) were determined by rate nephelometry using the Beckman Immunochemistry System Analyzer II (Beckman Instruments, Brea, CA). Circulating immune complexes were measured by conglutinin binding, and modified Clq binding assays. 21 CELLULAR IMMUNE EVALVA TION

Aqueous tear deficiency patients and controls enrolled in this study were examined for blood lymphocyte and natural killer cell deviations which have previously been reported to be associated with acute 13 ·22 and persistent EBV infection, 23 •24 and/or Sjogren's syndrome. 1•14- 16 •25 •26 Lymphocyte surface antigen detection was performed on peripheral blood mononuclear cells from patients and controls using heparinized venous blood. Single- and twocolor direct immunofluorescence was performed by staining the cells with fluorescein-isothiocyanate or phycoerythrin-conjugated murine monoclonal antibodies (Coulter Immunology, Hialeah, FL) against a variety of mononuclear cell surface antigens (Table 2). 27 Flow cytometry of stained cells was performed on an Epics C Cell Sorter (Coulter Electronics, Hialeah, FL) equipped with a 488-nm argon laser. Fluorescence of each cytochrome was determined by placing an appropriate filter over the fluorescence detector. Single- or dual-stained cells in a sample were expressed as a percentage of the total lymphocyte count using Quadrastat software (Coulter Immunology, Hialeah, FL). Evaluation of natural killer cell function was initiated at approximately halfway through the study, and as a result was not performed on all subjects. Natural killer cell function was evaluated by determining cytotoxicity against natural killer-sensitive erythroleukemic cells (K 562) using a whole blood chromium release assay as previously described. 28 Heparinized peripheral whole blood was mixed with Cr51 target cells at four different dilutions (0.25, 0.5, 1.0, and 2.0 X 106 ) in wells of a flat-bottom tissue culture tray. After 4 hours of incubation, the cellfree supernatant was removed and counted on a gamma counter (LKB Instruments, Rockville, MD). Controls to determine spontaneous and total Cr51 release were used. The percentage of cytotoxicity at the four different target/

Table 2. Antibodies Used for lmmunotyping of Blood Mononuclear Cells Antibody* B-cell marker B1 (CD20) T-cell markers T1 (CD5) T4 (CD4) T8 (COB) T11 (C02) 2H4 (CD45R) Natural killer cells NKH1 HLA class II 12

Primary Specificities Pre-B, immature, virginal, and secretory B cells All T-lymphocytes, certain B-cells Helper /inducer mature T-cells Cytotoxic/suppressor mature T-cells Sheep erythrocyte receptor, found on 95% of thymocytes 40% of peripheral lymphocytes, suppressor inducer function Mononuclear cells with natural killer activity HLA-DR-bearing cells (activated Tcells, most B-cells, macrophages)

* Abbreviations are the names of the monoclonal antibodies provided by the manufacturer (Coulter Immunology, Hialeah, FL). The CD number in parenthesis denotes the cluster-designate grouping of mononuclear cell antigens. 27

effector cell ratios, and the number ofNKH 1-labeled cells per unit of blood were used to calculate the percent cytotoxicity at a target to effector cell ratio of 1: 1. The maximal number of target cells lysed by each natural killer (NKHl antigen-positive) cell during the assay (kinetic lytic units) also was determined. EVALUATION OF GLANDULAR TISSUE

Palpebral lobe lacrimal gland biopsies were performed in six ATD patients with Sjogren's syndrome by a previously reported surgical technique. 29 The tissue was divided; a portion was placed in formaldehyde for light microscopy (6 cases), another portion was fixed in glutaraldehyde for transmission electron microscopy (3 cases), and the remainder was frozen in liquid nitrogen, and embedded in OCT (Tissue Tek, Napersville, IL). Shortly after death, control lacrimal glands for immunohistochemistry were obtained from four cadavers (20-60 years of age) with no history of ocular disease. Five-micron sections were cut from the frozen blocks, fixed in cold acetone, and frozen at -80° C in airtight containers. Each lacrimal gland specimen was tested for the presence ofEBV antigens using three monoclonal antibodies (R3.1, early diffuse; 2Ll0, membrane associated, and 5B 11 ), and CMV antigens using two monoclonal antibodies (2H2, early nuclear; 2F3, late nuclear). (EpsteinBarr virus and CMV antibodies were gifts of Dr. Gary Pearson, Georgetown University, Washington, DC.) An enhanced four-step immunoperoxidase technique as described by Gupta et al 30 was used. Epstein-Barr virus- and CMV-infected and negative cell lines were used as controls for immunoperoxidase staining. 315

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Table 3. Historic Information and Systemic Findings

Acute onset of ATD Duration < 2 yrs Primary Sjtlgren's syndrome Secondary Sjtlgren's syndrome History of infectious mononucleosis Fatigue Depression

Mild ATD (n = 18)

Severe ATD (n = 20)

No.(%)

No.(%)

7 (39)

11 (55) 13 (65)* 14 (70)t 2 (10) 3 (15) 4 (20) 3 (15)

6 (33) 2 (11) 1 (6) 0 (0} 0 (0) 0 (0)

ATD = aqueous tear deficiency. * P< 0.10. t P< 0.05.

BIOSTATISTICAL ANALYSIS

For antibody variables in this study, cutoffs for abnormally elevated levels of antibody based on laboratory means were established, and statistical tests were used to determine if the percentage of subjects with abnormal values were different in the control and disease groups. Since three study groups-controls, mild ATD, and severe ATD-were evaluated, we tested for trends across the groups in the proportion of cases having abnormal levels using Armitage's test for a linear trend in proportions. 31 We also used 3 X 2 contingency table analyses to compare the three groups. If a statistical significance in this test could not reasonably be accounted for by a trend, we compared the controls with any ATD, and the controls plus mild ATD against severe ATD to determine how the groups differed. When comparisons were made between two groups, Fisher's two-tailed exact test was used. Differences in the cell surface antigens and the natural killer cell function among the groups were evaluated using analysis of variance, or t tests on the original interval data. Kruskal-Wallis or the Mann-Whitney U tests were used when parametric assumptions were not met. Logistic regression 32 was used to construct multivariate models differentiating disease severity groups.

RESULTS PATIENT CHARACTERISTICS

We studied 38 patients with ATD, and 17 controls. Based on our severity grading system (Table 1), 20 patients were classified as having severe ATD, and 18 as having mild ATD. No differences in demographic features including age (mean and median), sex, and race were noted between the study groups. Historical information and systemic findings of ATD patients are noted in Table 3. A greater, but not statistically significant, percentage of severe ATD patients compared with the mild ATD group had a history of acute onset of ocular irritation (> 3-month period), and had symptoms of ocular irritation of less 316

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than 2 years' duration. Sjogren's syndrome was diagnosed in a greater proportion of severe (80%) than mild ( 18%) ATD patients (P < 0.001). Symptoms of fatigue and depression were reported only by severe ATD patients. These data suggest that differences exist in disease characteristics between the mild and severe ATD patients. A greater percentage of severe than mild patients had ATD of shorter duration and more acute onset, constitutional symptoms, and systemic disease (Sjogren's syndrome). VIRAL SEROLOGY

Antibody titers to EBV, CMV, and rubella from ATD patients (stratified by disease severity) and controls are presented in Figure 1. All ATD patients and controls had detectable antibody titers(~ 1:20) to EBV nuclear antigen. Epstein-Barr virus nuclear antigen titers were similar (P, not significant) between ATD patients and controls with no ATD or control patient having a titer greater than 1: 160. Statistically significant positive linear trends in the percentage of subjects with EBV viral capsid lgG titers of at least 1:640 (P < 0.05) and early antigen titers greater than 1: 160 (P < 0.00 l) or elevation of both viral capsid and early antigen titers (P < 0.05) were noted from the controls to severe ATD patients. Rubella titers were similar in the ATD patients and controls. A greater percentage of severe ATD patients had CMV titers greater than 2.6 enzyme immunoassay units (P < 0.02) compared with the mild and control groups. There was no correlation between EBV viral capsid or early antigen titers with rubella (r 2 = 0.016 and 0.00061, respectively) or CMV (~ = 0.024 and 0.01, respectively) titers. The viral serology results indicate that antibodies to EBV capsid and early antigens were increased in ATD patients compared with controls, and were positively correlated with disease severity. No correlation was noted between EBV and rubella or CMV titers suggesting that the elevated EBV antibodies in ATD patients were not due to nonspecific polyclonal B-cell activation. HUMORAL IMMUNE STUDIES

The results of autoantibody testing are noted in Table 4. There was a significantly increased frequency of antinuclear antibody (P < 0.001 ), rheumatoid factor (P < 0.001 ), and Sjogren's syndrome antibody A (P < 0.05) positivity from the controls to mild and sevete ATD patients. Sjogren's syndrome antibody Band nDNA antibodies were not detected in any subject. There was no difference in the serum IgA and lgM levels between the ATD and control groups. A positive trend in the percentage of patients with serum lgG greater than 1300 mg (P = 0.05) and immune complexes (P < 0.05) was noted from the control to severe ATD groups. These results indicate that serum autoantibodies, elevated serum lgG, and immune complexes were present in a greater percentage of ATD patients than controls and their presence correlates with disease severity.

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4

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Fig 1. Serum antibody titers (lgG) to Epstein-Barr virus (EBV) capsid antigen (top left), EBV early antigens (D+R) (bottom left), cytomegalovirus (CMV) (top right), and rubella virus (bottom right) for each study population. Epstein-Barr virus titers are expressed as the maximum serum titration at which immunofluorescence was detected. Cytomegalovirus and rubella virus titers are expressed as ELISA units. Titers above the line in each figure were used for statistical comparison. These cut-off values were based on predetermined laboratory means for each assay and represent titers of: EBV viral capsid IgG > 1:640, EBV early antigen> 1:160, CMV > 2.5 ELISA units, rubella virus> 0.7 ELISA units.

STUDIES OF CELL-MEDIATED IMMUNITY

One patient with severe ATD had anti-lymphocyte antibodies which produced uninterpretable flow cytometry results on three separate specimens. This patient was excluded from the figures and statistical analysis. The results of peripheral blood mononuclear cell immunotyping are presented in Table 5. An apparent reduction in number of total peripheral blood mononuclear cells (P = 0.092) and T-helper (CD4) cells (P = 0.063) was noted in the severe ATD group compared with the mild ATD and control groups. The average number and percentage of T -cells (C02), T -suppressor/cytotoxic (CDS), and B-cells (CD20) were similar in all groups. The average T-cell helper/suppressor (CD4/CD8) ratios were similar in the ATD and control groups; however, a greater variability in the ratios (P::; 0.005) was noted in the ATD patients compared with controls (i.e., a greater percentage of ATD patients than controls had ratios > 3 or < 1, Fig 2, [top left]). 33 All six patients with a CD4/CD8 ratio of less than 1.0 were HIV 1 antibody-negative. T-helper lymphocytes bearing 2H4 antigens (T4+2H4+) have been reported to be involved in induction of suppressor cells, 34 and have been found to be reduced in Sjogren's syndrome

Table 4. Humoral Immune Evaluation

Antinuclear antibody Rheumatoid factor Sjogren's syndrome antibody At lgG > 1300 mg/dl Immune complexes+ C1Q CB

Control (n = 17)

Mild ATD (n = 18)

Severe ATD (n = 20)

No.(%)

No.(%)

No.(%)

1 (6) 0 (0)

7 (39) 6 (33)

16 (80)* 13 (65)*

0 (0) 2 (12)

3 (17) 5 (28)

6 (30)* 8 (40)*

0 (0) 0 (0)

2 (11) 0 (0)

9 (45)* 3 (15)

ATD = aqueous tear deficiency; CB = conglutinin binding. * P,;; 0.05 by test of trend. t SjOgren's syndrome antibody B was not detected in any group. + Circulating immune complexes were detected by two methods-C1 Q binding and conglutinin binding.

patients. 26 Figure 2, bottom left, shows the percentage of T4+ 2H4+ cells in all groups. A greater percentage of ATD patients had less than 10% T4+2H4+ cells compared with 317

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Table 5. Peripheral Blood Mononuclear Cell Analysis Mean± SO Group

PBMN (cell/mm 3)

T4 (cellsjmm 3}

T4 (%)

T8 (%}

T11 (%)

Severe ATD (n = 19) Mild ATO (n = 18) Control (n = 17)

1579 ± 764 2235 ± 1071 2071 ± 778

737 ± 412* 1148 ± 753 1017 ± 473

50± 15 49 ± 13 51± 6

29 ± 7 30 ± 7 28 ± 6

80 ± 9 80 ± 5 77 ± 16

T4 + 2H4+ (%)

T4/T8 Ratio

16 ± 9 13 ± 7 19 ± 8

2.0 ± 1.0 1.8±0.9 1.9±0.5

SO = standard deviation; PBMN = peripheral blood mononuclear cells; ATO = aqueous tear deficiency. * p = 0.063.

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left, suppressor inducer Fig 2. Results of peripheral blood mononuclear cell immunotyping. Top left, T-cell helper/suppressor ratios (T4/T8); bottom B-cells bearing T-cell right, bottom ; (T8+12+/T8+) antigens HLA-DR bearing cells T helper cells (T4+2H4+); top right, fraction ofCD8+ (T8+) than 3. Right, the greater or I than less ratios (CD4/CD8) or helper/suppress T indicate lines the left, Top +). +Bl (Tl antigens surface Tl) or (CD5 comparison. 33 statistical for used were cut-offs These value. control of 90% below lines indicate above 90% of control value. Bottom left, the lines indicate

controls (P 5, 0.05); however, no trend with disease severity was observed. A marked increase in HLA class II antigen bearing Tsuppressor/cytotoxic cells (T8+12+/T8 +) was noted in the severe ATD group compared with the mild ATD and control groups (P 5, 0.001, Fig 2, (top right). The percentage of circulating B-cells bearing a T -cell (T 1 or CDS)associated antigen (T 1+ B 1+) has recently been reported 318

25 to be increased in Sjogren's syndrome patients. A greater, our ATD of but not statistically significant, percentage (>9%) percentage elevated an had patients than control of T1 + B cells (P 5, 0.1, Fig 2, bottom right). The percentage of peripheral blood mononuclea r cells bearing natural killer cell-associated surface antigens was similar in all groups. Linear trends of reduced natural killer cell cytotoxicity (P < 0.05, Fig 3), and reductions

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in the maximal number of cells lysed by each natural killer cell-associated surface antigen (kinetic lytic units, P < 0.03) were found from controls to severe ATD patients. Compared with the control group, ATD patients showed a greater variability in T-helper/suppressor cell ratios, a reduced percentage ofT-helper cells involved in inducing suppression (T4+2H4+), and increased percentage of B-cells bearing a T-cell associated antigen (T I+ B I+). Furthermore, a reduction in total and T-helper lymphocytes, reduced natural killer cell cytotoxicity and kinetic lytic units, and an increased percentage of HLADR + CDS lymphocytes was observed in severe ATD patients. MULTIVARIATE ANALYSIS

Logistic regression was used to determine which variables best divided ATD patients into mild and severe groups. Three variables, elevated percentage ofHLA-DR + CDS lymphocytes, Sjogren's syndrome, and elevated EBV early antigen titers were found to differentiate between mild and severe ATD cases. The model estimated odds of being in the severe ATD category were increased 7. 7 times for subjects with an elevated percentage of HLADR+ CDS lymphocytes (95% confidence interval, 1.540.6), 6.5 times for subjects with Sjogren's syndrome (95% confidence interval, 1.4-29.2), and 3.9 times for subjects with elevated EBV early antigen titers (95% confidence interval, 1.0-I7 .4 ). It should be noted that although the TSI2/TS ratio is important for separating mild and severe ATD, a linear trend from controls to severe ATD was not observed. LACRIMAL GLAND EVALUATION

Palpebral lobe lacrimal gland biopsies were performed in six severe ATD patients with Sjogren's syndrome. Moderate-to-intense lymphocytic infiltration was noted in all six lacrimal glands from ATD patients. This was either diffuse (4 glands) or sectorial (2 glands). No herpesviruses were visualized by transmission electron microscopy in the three biopsies examined. Epstein-Barr virus antigens were detected in two biopsies. Scattered stained cells were noted with anti-EBV monoclonal antibodies R3.I and 2Ll 0 in both biopsies and 5B II antibody in only one (Fig 4, top). Epstein-Barr virus antigen containing cells were found to bear B-lymphocyte-associated surface antigens on serial sections (results not presented). Staining with both anti-CMV monoclonal antibodies was noted at similar locations on serial sections in one of the EBV antigen-positive biopsies. Scattered staining with CMV antibodies was noted in one gland negative for EBV antigens. Staining was not observed in the four control glands with EBV or CMV antibodies (Fig 4, bottom).

DISCUSSION The finding of elevated antibodies to EBV, a member of the gammaherpesvirus family, in ATD patients com-

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Fig 3. Results of natural killer cell cytotoxicity studies. The line indicates a percent cytotoxicity of 12% used for statistical comparison.

pared with a control group with normal tear production supports our hypothesis that there is an association between EBV infection and development of ATD. Antibody titers to rubella and CMV, viruses to which most adults in the United States have been exposed, 35 •36 were found to have no correlation with EBV titers. This suggests that the elevation of antibodies to EBV in ATD patients is a specific response rather than the result of nonspecific polyclonal B-cell activation. The greatest elevation of EBV titers was observed in severe ATD patients. The severe ATD group was composed predominantly of patients with Sjogren's syndrome, and compared with the mild group, the severe ATD patients had a history of more acute onset and shorter duration ATD. They also had greater immunologic dysfunction. A multivariate analysis of disease parameters showed a significant correlation between severe ATD and elevated EBV early antigen titers, Sjogren's syndrome, and an increased percentage ofHLA class II antigen bearing CDS+ cells. A significantly smaller percentage of patientsin the mild ATD group had elevated EBV serology, and it is possible that EBV infection is a risk factor for ATD in those mild ATD patients with elevated EBV antibodies. The lower EBV antibody titers in the mild ATD group may reflect differences in disease characteristics noted between the mild and severe groups such as duration of ATD or immune dysfunction. Alternatively, mechanisms unrelated to EBV infection may be responsible for development of ATD in the majority of patients with mild disease. Epstein-Barr virus is a ubiquitous herpesvirus recognized as the cause of infectious mononucleosis. 37 It also has been implicated in the pathogenesis of several different human neoplasms including Burkitt's lymphoma, nasopharyngeal carcinoma, thymic carcinoma, and B-cell lymphomas in immunodeficient patients. 20 A characteristic serum antibody response to EBV antigens during infectious mononucleosis has been described. 19 By the time clinical symptoms of infectious mononucleosis have developed, both lgM and lgG antibodies to viral capsids have reached their peak. After recovery from mononu3I9

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Fig 4. Top, lacrimal gland biopsy performed in a 52-yearold woman 9 months after acute onset of severe aqueous tear deficiency stained with monoclonal antibodies to EBV early diffuse antigen (R 3.1) as described in the Patients and Methods section (original magnification, X200). This patient had filamentary keratitis, and conjunctival squamous metaplasia in the exposure and non-exposure zones. Laboratory analysis at the time of the biopsy was: serum lgG = 31 10 mgjdl, elevated circulating immune complexes, SS-A 1:128, rheumatoid factor 1:1075, ANA 1:5120, EBV VCA lgG 1:2560, EBV IgM VCA IgM I:40, EBV EA (D+R) lgG 1:2560, EBV EBNA 1:160, CMV = 3.0 EU. Anti-lymphocyte antibodies produced uninterpretable flow cytometry results on three separate specimens. Bottom, lack of staining with R 3.1 antibody in a control lacrimal gland (original magnification, X200).

cleosis, viral capsid lgM antibodies disappear; however, anti-viral capsid lgG persists at low levels throughout life. Antibody to early antigens which are indicative of viral replication decrease to low, or non-detectable levels in most individuals after the disease resolves. 19 Antibodies to Epstein-Barr nuclear antigen appear weeks to months 320

after the onset of infectious mononucleosis, persist for life, and provide serologic evidence of past EBV infection. Specific patterns of elevated antibodies to EBV antigens have been found in patients with EBV-associated diseases: Burkitt's lymphoma (viral capsid lgG, early antigen restricted component), nasopharyngeal carcinoma (viral

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capsid lgG and lgA, early antigen diffuse component), recurrent parotitis in children (viral capsid lgG and IgA, early antigen restricted component), chronic fatigue syndrome (viral capsid IgG, early antigen). 23 •38 Antibody titers have been found to have a positive correlation with disease activity of EBY-associated neoplasia. 39 Aqueous tear deficiency patients in this study had a pattern of detectable anti-Epstein-Barr nuclear antigen with elevated early antigens viral capsid antibodies which is consistent with chronic or reactivated EBY infection. 38 We have previously reported the onset of Sjogren's syndrome shortly after serologically confirmed infectious mononucleosis. 2 All three ATD patients with a history of infectious mononucleosis in our current study noted onset of ocular irritation within several months after this infection. Another ATD patient (Fig 4, top) who experienced acute onset of ATD 9 months before our initial evaluation was found to have elevated IgM viral capsid antibodies, suggesting a recent primary EBY infection. These cases of ATD after primary EBY infection indicate that EBY infection may be an initiating event for the development of ATD in a subset of ATD patients. Most ATD patients with elevated EBY antibodies in our study had no history of infectious mononucleosis, and from their pattern of EBY serology it is not possible to determine whether infection persisted after an unrecognized primary exposure, or if a latent virus reactivated after a past infection which resolved normally. We have recently found that the lymphocytic infiltrate within the lacrimal gland of primary Sjogren's syndrome patients represents a B-lymphocyte proliferation which is occasionally monoclonal, and is accompanied by increased numbers of helper T-cells (unpublished data). Immunoglobulin gene rearrangements indicating clonal expansion of B-cells have been detected in several £BYassociated diseases including Hodgkin's disease, 40 B-cell lymphomas, 41 and Castleman's disease. 42 They also have been detected in salivary gland lymphoepitheliallesions of Sjogren's syndrome patients. 43 Epstein-Barr virus is a well-recognized stimulus for B-lymphoproliferation. 44 The cause of lacrimal gland B-cell proliferation in Sjogren's syndrome patients has not been established. We hypothesize that the B-cell proliferation of the lacrimal gland of ATD patients is initiated by EBY infection of glandular B-cells. The elevated EBY. antibodies noted in this study provide indirect evidence of this infection. Our hypothesis is supported by the detection of £BYassociated antigens in glandular B-cells of two ATD patients evaluated in this study. The immunologic detection of viral antigens within the lacrimal gland by itself, however, is insufficient proof ofEBY infection of the lacrimal gland since the monoclonal antibodies used in this study could be detecting cross-reactive tissue epitopes. Antibodies from patients with infectious mononucleosis have been demonstrated to react with cytoskeletal antigens such as cytokeratin and vimentin, both of which are present within the lacrimal gland. 12 The data, however, weighs against this. First, several different EBY antigens were detected. Second, no antigens were recognized by the viral monoclonal antibodies in the control lacrimal gland tissue

which contains the same cytoskeletal antigens. To confirm the theory that the B-lymphocytic infiltration of lacrimal glands of Sjogren's syndrome patients is induced by EBY, viral genomes or antigens and mRNA transcripts associated with EBY induced B-cell proliferation45 must be demonstrated within the lacrimal gland. Eighty-percent of the severe A TD patients in our study had Sjogren's syndrome. This indicates that most patients with severe ATD using our classification system (Table I) have an immune-mediated lacrimal gland dysfunction. The mechanism responsible for inciting lymphocytic infiltration and dysfunction of salivary glands in Sjogren's syndrome also is probably responsible for the lacrimal gland pathology in this disease. Fox and associates9 have recently demonstrated EBY antigens and DNA within minor labial and parotid salivary gland tissue, and saliva of patients with primary Sjogren's syndrome. They postulated that the lymphocytic infiltration of salivary glands in primary Sjogren syndrome patients is related to reactivated EBY infection within the glands of predisposed individuals. One predisposing factor may be HLA type since there is an increased prevalence of HLA DR-3 and DW-3 antigens in primary Sjogren's syndrome patients. 46 In order for a viral-infected cell to be recognized by the immune system, viral antigens must be presented toThelper cells in association with HLA-DR antigens by antigen presenting cells. 47 It is possible that immune recognition ofEBY-infected cells and subsequent suppression of this .infection are impaired in patients with certain HLA-DR antigens such as those associated with Sjogren's syndrome. Further investigation of the relationship of HLA-DR type and suppression of EBY infection may provide valuable insight into the molecular mechanisms of genetic predisposition to Sjogren's syndrome. Elevated levels of serum immunoglobulins and autoantibodies are hallmarks of Sjogren's syndrome. As in Sjogren's syndrome, patients with acute infectious mononucleosis have evidence ofB-cell activation, including elevated serum immunoglobulins and autoantibodies. These findings in mononucleosis patients are related to EBY stimulation of immunoglobulin production by Bcells.22 Cellular immune effectors rapidly develop in patients with infectious mononucleosis which suppress £BYinduced B-cell activation. 22 Although the mechanisms involved in this suppression are not completely understood, they appear to involve T-lymphocytes and natural killer cellsY Defective immune suppression of EBY-induced B-cell proliferation ~nd immunoglobulin production has been found in primary Sjogren's syndrome 48 as well as several immune diseases associated with Sjogren's syndrome49 such as rheumatoid arthritis, systemic lupus erythematosis, and acquired immune deficiency syndrome (AIDS).49-52 In our current study, we observed alterations of several cellular immune parameters in ATD patients. The greatest reductions in total and T-helper (CD4) lymphocyte cell counts and natural killer cell function were noted in severe ATD patients, the group which had the greatest elevation of EBY serology and proportion of Sjogren's syndrome. Epstein-Barr virus-induced B-cell proliferative disorders 321

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occur with increased frequency in immunosuppressed transplant recipients, and AIDS patients. 44 Patients with these immunologic diseases have cellular immune abnormalities similar to those observed in our severe ATO group. Both EBV and human herpes type 6 genomic sequences have been detected in B-cell lymphomas from Sjogren's syndrome patients. 9 •10 It is possible that depressed cellular immune function in our severe ATO patients, as in transplant recipients and HIV-infected patients, is a factor predisposing to persistent EBV infection. Alternatively, EBV infection may be inducing these cellular immune deviations. Lysis ofT-helper lymphocytes after infection by a lymphotrophic herpesvirus, human herpes type 6 virus, has been reported. 53 Epstein-Barr virus appears to be capable of infecting T-lymphocytes, 54 and if this occurs in ATO patients, it could be responsible for cytolysis and reduced lymphocyte cell counts. Further research is necessary to elucidate the relationship between cellular immune dysfunction and EBV infection in ATO patients. An increased proportion of CD8+ lymphocytes expressing HLA-DR antigen was found to be a highly significant risk factor for severe ATO. HLA-DR antigens are normally expressed on B-lymphocytes and antigen-presenting cells such as macrophages. Inappropriate expression of HLA-DR antigens on other types of cells, such as the CD8+ lymphocytes, can be induced by class II modulating factors, principally interferon-gamma. 5 5 Viral infection stimulates cells to produce interferon-gamma. An increased percentage ofHLA-DR bearing CD8+ cells was observed by Franco and associates24 in patients with severe chronic EBV infection. The inappropriate HLA-DR expression on CD8+ cells noted in our study may be related to increased interferon-gamma production in response to EBV infection. An elevated percentage ofHLADR bearing CD8+ cells may prove to be a useful immune marker of persistent viral infection in ATO patients in future studies. Cytomegalovirus is a herpesvirus-like EBV that is capable of causing persistent or recurrent infections after primary infection. The majority of adults have serologic evidence of previous CMV infection. 36 The kinetics of CMV antibody production after primary infection are not as well defined as EBV; however, elevated anti-CMV lgG antibodies have been detected in immunocompromised patients with chronic or recurrent CMV infection. 36 Elevated CMV antibody titers and CMV antigens in lacrimal gland biopsies were found only in severe ATO patients in this study. These findings may represent chronic or recurrent CMV infection in the severe ATO patients resulting from immune dysfunction. Alternately, CMV may play a direct role in the pathogenesis of lacrimal gland dysfunction in these patients. The relationship of this virus, as well as EBV, to development of ATD requires future evaluation. In this and previous studies, ATO has been noted to occur primarily in postmenopausal women. Sex hormones may have modulating effects on the immune system. Increased cervical shedding of CMV has been observed at different stages of pregnancy, and may reflect changes in 322

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levels of circulating hormones. 36 As with the other risk factors for ATO discussed previously, elucidation of the molecular basis of the sex predisposition for ATO requires further investigation. If future research more clearly establishes a relationship between persistent EBV (or other herpes viral infection) and development of ATD, therapy of ATO in the subset of patients with evidence of viral infection could be directed toward treatment of the infection with either antiviral agents or immune system augmentation.

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