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Elevated tear interleukin-6 levels in patients with Sjögren syndrome
Elevated Tear Interleukin-6 Levels in Patients with Sjo¨gren Syndrome Moshe Tishler, MD,1 Ilana Yaron, MSci,1 Orna Geyer, MD,2 Idit Shirazi, PhD,1 Eva Naftaliev, MD,2 Michael Yaron, MD1 Objective: The purpose of the study was to investigate interleukin-6 (IL-6) levels in the tear fluid and sera of patients with Sjo¨gren syndrome (SS). Participants: Twelve patients with primary SS and 12 normal control subjects participated. Intervention: Tear fluid and sera were obtained from the study and the control groups. Evaluation of tear fluid and sera IL-6 levels was done by using a quantitative enzyme-linked immunosorbent assay kit. All assays were carried out blindly with respect to diagnosis. Main Outcome Measures: Tear fluid IL-6 levels were measured. Results: The mean concentration (⫾ standard error) of IL-6 in the tears of patients with SS was elevated significantly compared to that of normal control subjects (88.6 ⫾ 16.2 vs. 42.1 ⫾ 10.6 pg/ml; P ⬍ 0.05). No significant differences were noted in the serum IL-6 levels between the two groups. A significant correlation (r ⫽ 0.742, P ⫽ 0.006) was found between tear fluid IL-6 levels and the focus score of lip biopsy specimens in patients with SS. Conclusion: Tear fluid IL-6 levels may serve as an important marker for tear gland involvement in SS. Ophthalmology 1998;105:2327–2329 Dry eyes are an important manifestation in Sjo¨gren syndrome (SS) but are neither diagnostic nor specific. Keratoconjunctivitis sicca (KCS) is not specific for SS; it has been found in more than 15% of persons older than 60 years of age1 and in a variety of immunologic disturbances such as human immunodeficiency virus infection, graft-versus-host disease, and human T-cell leukemia/lymphoma virus-I infection.2 In SS, the dry eye appears to be caused primarily by reduced tear secretion from the main and accessory lacrimal glands, although both mucin insufficiency and Meibomian gland dysfunction may play a role.3 The precise mechanisms responsible for the reduced tear production in SS are not known. Classically, it has been attributed to a progressive lymphocytic infiltration composed predominantly of B and CD4 lymphocytes, which causes destruction of acinar and ductal epithelial cells.4 Studies of lacrimal tissue from patients with SS have shown an elevated expression of mRNA to interleukin-1 (IL-1), interleukin-6 (IL-6), and ␥-interferon (␥-IFN) as well as elevated levels of various adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1).5 Recently, high tear fluid plasmin activity was detected in patients with SS, suggesting that proteolytic activity may play a role in the physiopathology Originally received: March 16, 1998. Revision accepted: July 28, 1998. Manuscript no. 98141. 1 Department of Rheumatology, Tel Aviv Souraski Medical Center and the Tel Aviv University Sackler School of Medicine, Tel Aviv, Israel. 2 Department of Ophthalmology, Tel Aviv Souraski Medical Center and the Tel Aviv University Sackler School of Medicine, Tel Aviv, Israel. Reprint requests to Moshe Tishler, MD, Department of Rheumatology, Tel Aviv Souraski Medical Center, 6 Weizmann St., Tel Aviv 64239, Israel.
in these patients.6 These findings are similar to those reported in salivary gland biopsy specimens of patients with SS.7,8 The active local production of cytokines by the glandular epithelial cells, expressed by elevated salivary IL-6 levels, prompted us to explore the possibility that such increased levels also could be found in the tears of patients with SS and may serve as a marker of local inflammation.
Patients and Methods Twelve patients with primary SS (10 women and 2 men), followed at the SS Clinic, Department of Rheumatology, Tel Aviv Medical Center, were randomly included in the study. Diagnosis of SS was made according to the newly proposed criteria of the European Community study group.9 Briefly, the existence of at least four of the six following items is indicative of primary SS: (1) ocular symptoms, (2) oral symptoms, (3) evidence of KCS, (4) focal sialoadenitis by minor salivary biopsy, (5) evidence of salivary gland involvement, and (6) presence of autoantibodies. A prerequisite for diagnosis was the performing of a lip biopsy, the results of which showed a focus score of 1 or greater. None of the patients had taken corticosteroids, hydroxychloroquine, or methotrexate before collection of tears nor had any apparent eye infection. Results obtained from these patients were compared to data of 12 healthy patients referred for an ambulatory cataract extraction. The clinical and serologic data of patients are presented in Table 1. Patients and control subjects underwent a complete ophthalmologic examination. This included standard biomicroscopy of the anterior segment of the eye with special attention to tear meniscus height, conjunctival or corneal mucus discharge, and the presence of filaments. Intraocular pressure was measured with the Goldmann applanation tonometer. Schirmer’s test was done without anesthesia to the patient using commercial Schirmer test strips placed in the lower temporal fornices of both eyes for 5 minutes.
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Ophthalmology Volume 105, Number 12, December 1998 Table 1. Clinical and Serologic Data of the Study Groups
Age (mean ⫾ SD) (yrs) Sex (M/F) RF ⬎ 1:160 ANF ⬎ 1:80 Anti-Ro antibodies Anti-La antibodies Schirmers’s test (mean ⫾ SD)
SS Patients (n ⴝ 12)
Normal Controls (n ⴝ 12)
55.3 ⫾ 6.3 2/10 9 (75%) 8 (66%) 8 (66%) 4 (33%) 4.3 ⫾ 1.1
60.1 ⫾ 9.9 4/8 0 (0%) 0 (0%) 0 (0%) 0 (0%) 22 ⫾ 5
SS ⫽ Sjo¨gren syndrome; SD ⫽ standard deviation; RF ⫽ rheumatoid factor; ANF ⫽ antinuclear factor.
Interleukin-6 Assays
Figure 2. Correlation of tear fluid interleukin-6 levels and the focus score of lip biopsies in patients with Sjo¨gren syndrome.
Tear fluid and serum were obtained from patients at the same visit. Tear fluid was collected from the lower cul-de-sac using 5 l blunted-tip micropippets and immediately was frozen and stored at ⫺20° C until processed. The assay was carried out blindly with respect to diagnosis. Tear fluid and serum IL-6 levels were evaluated using a quantitative enzyme-linked immunosorbent assay kit (Quantikine High Sensitivity, R&D Systems Inc, Minneapolis, MN) with a threshold sensitivity of 0.095 pg/ml.
9.9 pg/ml). A significant correlation was found between tear fluid IL-6 levels of patients with SS and the focus score of lip biopsies (r ⫽ 0.742; P ⫽ 0.006) (Fig 2). No correlation could be found between tear fluid IL-6 concentrations and age, duration of disease, serum IL-6 levels, the presence of autoantibodies, or clinical disease manifestations.
Statistical Analysis
Discussion
The significance of differences between continuous variables was determined by using paired Students’ t test. Correlations between tear fluid IL-6 concentrations and various disease parameters were determined using the Pearson product moment correlation.
Results The concentrations of IL-6 in the tear fluid of the study groups are shown in Figure 1. The mean concentration (⫾ standard error) of IL-6 in the tears of patients with SS was 88.6 ⫾ 16.2 pg/ml compared to 42.1 ⫾ 10.6 pg/ml in normal control subjects (P ⬍ 0.05). No significant differences were noted in the serum IL-6 levels between patients with SS and control subjects (37 ⫾ 11.9 pg/ml vs. 25.1 ⫾
Figure 1. Tear fluid interleukin-6 levels in patients with Sjo¨gren syndrome and in control subjects.
2328
Primary SS is a multisystem autoimmune disorder characterized mainly by KCS and xerostomia. The current tests that establish the presence of KCS in patients suspected of having SS are the Schirmer’s test, tear film break-up time, and the rose– bengal staining of the ocular surface.10 A recent comprehensive study has shown that with a limited number of clinical tests, there is a high probability that the common causes of ocular irritation can be diagnosed differentially.11 Nevertheless, the laboratory work-up still is incomplete. The tear fluid contains a large variety of substances, whose significance still remains obscure. Measurements of these components, lysozome, lactoferrin, ␣-antitrypsine, peroxidase, and 2-microglobulin, have been proposed as an aid for the diagnosis of SS.12,13 The results of our study show that patients with SS have increased levels of IL-6 in their tear fluid but not in their sera. These findings are complementary to those of an earlier study, which showed an increase in 2 microglobulin, a known marker of inflammation, in the tear fluid of 35 patients with SS.13 The local increase of IL-6 in the tear fluid also is in accordance with our recent findings and others, indicating that increased salivary IL-6 levels could be detected in the saliva of patients with SS.14,15 Furthermore, a positive correlation has been found between tear IL-6 levels and the severity of lymphocytic infiltration of minor salivary glands, similar to that found by us in salivary IL-6 levels. Because in both the lacrimal and the salivary glands CD4⫹ lymphocytic infiltration is characteristic in SS, it is conceivable that the infiltrating lymphocytes of the lacrimal glands are the source of IL-6 in the tear fluid of our patients with SS. One might speculate that the elevated tear fluid IL-6 levels are a consequence of concentration because of the small volume of tears found in patients with SS.
Tishler et al 䡠 Tear IL-6 Levels in SS However, several arguments can be raised against this determination. Although small volumes of tear fluid are the hallmark of SS, concentrations of different tear fluid constituents vary. While concentrations of lactoferrin and lysozyme and peroxidase are reduced, increased concentrations of 2-microglobulin and ␣1 antitrypsine are found in the tear fluid of patients with SS. Furthermore, no correlation between IL-6 tear fluid levels and age, disease duration, or Schirmer’s test could be found in our group of patients as in another study concerning 2-microglobulin levels.13 Interleukin-6, which is a multifactorial cytokine, is involved in T-cell differentiation, stem cell proliferation, and acutephase protein production.16 The concept that quite a simple noninvasive method, which does not require specific equipment, can help in the diagnosis of SS is attractive. To the best of our knowledge, this is the first study that shows elevated IL-6 levels in the tear fluid of patients with SS. Because the rise in IL-6 might be just a marker of CD4⫹ lymphocytic accumulation, further studies with larger numbers of patients, which will include other autoimmune disorders and nonimmune-mediated inflammatory eye diseases, are needed to determine the specificity of this finding for SS.
References 1. Jacobsson LT, Axell TE, Hansen BU, et al. Dry eyes or mouth-an epidemiological study in Swedish adults, with special reference to primary Sjo¨gren’s syndrome. J Autoimmun 1989;2:521–7. 2. Prause JU. Dry Eye in Sjo¨gren’s Syndrome-State of the Art. Homma M, Sugai S, Tojo T, Miyasaka N, Akuzuki M, eds. Amsterdam: Kugler Publications, 1994;5– 8. 3. A comprehensive guide. Lemp MA, Marquardt R, eds. The Dry Eye: Berlin; New York: Springer–Verlag, 1992;65–99. 4. Pepose JS, Akata RF, Pflugfelder SC, Voigt W. Mononuclear cell phenotypes and immunoglobulin gene rearrangements in
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
lacrimal gland biopsies for patients with Sjo¨gren’s syndrome. Ophthalmology 1990;97:1599 – 605. Saito I, Terauchi K, Shimuta M, et al. Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjo¨gren’s syndrome. J Clin Lab Anal 1993;7:180 –7. Virtanen T, Konttinen YT, Honkanen N, et al. Tear fluid plasmin activity of dry eye patients with Sjo¨gren’s syndrome. Acta Ophthalmol Scand 1997;75:137– 41. Fox RI, Kang HI, Ando D, et al. Cytokine mRNA expression in salivary gland biopsies of Sjo¨gren’s syndrome. J Immunol 1994;152:5532–9. Boumba D, Skopouli FN, Moutsopoulos HM. Cytokine mRNA expression in the labial salivary gland tissues from patients with primary Sjo¨gren’s syndrome. Br J Rheumatol 1995;34:326 –33. Vitali C, Bombardieri S, Moutsopoulos HM, et al. Preliminary criteria for the classification of Sjo¨gren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 1993;36:340 –7. Prause JU. Clinical ophthalmological tests for the diagnosis of keratoconjunctivitis sicca. Clin Exp Rheumatol 1989;7: 141– 4. Pflugfelder SC, Tseng SCG, Sanabria O, et al. Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation. Cornea 1998;17:38 –56. Markusse HM, van Haeringen NJ, Swaak AJ, et al. Tear fluid analysis in primary Sjo¨gren’s syndrome. Clin Exp Rheumatol 1993;11:175– 8. Markusse HM, Huysen JC, Nieuwenhuys EJ, Swaak AJ. Beta 2-microglobulin in tear fluid from patients with primary Sjo¨gren’s syndrome. Ann Rheum Dis 1992;51:503–5. Tishler M, Yaron I, Shirazi I, Yaron M. Saliva: an additional diagnostic tool in Sjo¨gren’s syndrome. Semin Arthritis Rheum 1997;27:173–9. Grisius MM, Bermudez DK, Fox PC. Salivary and serum interleukin 6 in primary Sjo¨gren’s syndrome. J Rheumatol 1997;24:1089 –91. Brach MA, Hermann F. Interleukin 6: presence and future. Int J Clin Lab Res 1992;22:143–51.
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in these patients.6 These findings are similar to those reported in salivary gland biopsy specimens of patients with SS.7,8 The active local production of cytokines by the glandular epithelial cells, expressed by elevated salivary IL-6 levels, prompted us to explore the possibility that such increased levels also could be found in the tears of patients with SS and may serve as a marker of local inflammation.
Patients and Methods Twelve patients with primary SS (10 women and 2 men), followed at the SS Clinic, Department of Rheumatology, Tel Aviv Medical Center, were randomly included in the study. Diagnosis of SS was made according to the newly proposed criteria of the European Community study group.9 Briefly, the existence of at least four of the six following items is indicative of primary SS: (1) ocular symptoms, (2) oral symptoms, (3) evidence of KCS, (4) focal sialoadenitis by minor salivary biopsy, (5) evidence of salivary gland involvement, and (6) presence of autoantibodies. A prerequisite for diagnosis was the performing of a lip biopsy, the results of which showed a focus score of 1 or greater. None of the patients had taken corticosteroids, hydroxychloroquine, or methotrexate before collection of tears nor had any apparent eye infection. Results obtained from these patients were compared to data of 12 healthy patients referred for an ambulatory cataract extraction. The clinical and serologic data of patients are presented in Table 1. Patients and control subjects underwent a complete ophthalmologic examination. This included standard biomicroscopy of the anterior segment of the eye with special attention to tear meniscus height, conjunctival or corneal mucus discharge, and the presence of filaments. Intraocular pressure was measured with the Goldmann applanation tonometer. Schirmer’s test was done without anesthesia to the patient using commercial Schirmer test strips placed in the lower temporal fornices of both eyes for 5 minutes.
2327
Ophthalmology Volume 105, Number 12, December 1998 Table 1. Clinical and Serologic Data of the Study Groups
Age (mean ⫾ SD) (yrs) Sex (M/F) RF ⬎ 1:160 ANF ⬎ 1:80 Anti-Ro antibodies Anti-La antibodies Schirmers’s test (mean ⫾ SD)
SS Patients (n ⴝ 12)
Normal Controls (n ⴝ 12)
55.3 ⫾ 6.3 2/10 9 (75%) 8 (66%) 8 (66%) 4 (33%) 4.3 ⫾ 1.1
60.1 ⫾ 9.9 4/8 0 (0%) 0 (0%) 0 (0%) 0 (0%) 22 ⫾ 5
SS ⫽ Sjo¨gren syndrome; SD ⫽ standard deviation; RF ⫽ rheumatoid factor; ANF ⫽ antinuclear factor.
Interleukin-6 Assays
Figure 2. Correlation of tear fluid interleukin-6 levels and the focus score of lip biopsies in patients with Sjo¨gren syndrome.
Tear fluid and serum were obtained from patients at the same visit. Tear fluid was collected from the lower cul-de-sac using 5 l blunted-tip micropippets and immediately was frozen and stored at ⫺20° C until processed. The assay was carried out blindly with respect to diagnosis. Tear fluid and serum IL-6 levels were evaluated using a quantitative enzyme-linked immunosorbent assay kit (Quantikine High Sensitivity, R&D Systems Inc, Minneapolis, MN) with a threshold sensitivity of 0.095 pg/ml.
9.9 pg/ml). A significant correlation was found between tear fluid IL-6 levels of patients with SS and the focus score of lip biopsies (r ⫽ 0.742; P ⫽ 0.006) (Fig 2). No correlation could be found between tear fluid IL-6 concentrations and age, duration of disease, serum IL-6 levels, the presence of autoantibodies, or clinical disease manifestations.
Statistical Analysis
Discussion
The significance of differences between continuous variables was determined by using paired Students’ t test. Correlations between tear fluid IL-6 concentrations and various disease parameters were determined using the Pearson product moment correlation.
Results The concentrations of IL-6 in the tear fluid of the study groups are shown in Figure 1. The mean concentration (⫾ standard error) of IL-6 in the tears of patients with SS was 88.6 ⫾ 16.2 pg/ml compared to 42.1 ⫾ 10.6 pg/ml in normal control subjects (P ⬍ 0.05). No significant differences were noted in the serum IL-6 levels between patients with SS and control subjects (37 ⫾ 11.9 pg/ml vs. 25.1 ⫾
Figure 1. Tear fluid interleukin-6 levels in patients with Sjo¨gren syndrome and in control subjects.
2328
Primary SS is a multisystem autoimmune disorder characterized mainly by KCS and xerostomia. The current tests that establish the presence of KCS in patients suspected of having SS are the Schirmer’s test, tear film break-up time, and the rose– bengal staining of the ocular surface.10 A recent comprehensive study has shown that with a limited number of clinical tests, there is a high probability that the common causes of ocular irritation can be diagnosed differentially.11 Nevertheless, the laboratory work-up still is incomplete. The tear fluid contains a large variety of substances, whose significance still remains obscure. Measurements of these components, lysozome, lactoferrin, ␣-antitrypsine, peroxidase, and 2-microglobulin, have been proposed as an aid for the diagnosis of SS.12,13 The results of our study show that patients with SS have increased levels of IL-6 in their tear fluid but not in their sera. These findings are complementary to those of an earlier study, which showed an increase in 2 microglobulin, a known marker of inflammation, in the tear fluid of 35 patients with SS.13 The local increase of IL-6 in the tear fluid also is in accordance with our recent findings and others, indicating that increased salivary IL-6 levels could be detected in the saliva of patients with SS.14,15 Furthermore, a positive correlation has been found between tear IL-6 levels and the severity of lymphocytic infiltration of minor salivary glands, similar to that found by us in salivary IL-6 levels. Because in both the lacrimal and the salivary glands CD4⫹ lymphocytic infiltration is characteristic in SS, it is conceivable that the infiltrating lymphocytes of the lacrimal glands are the source of IL-6 in the tear fluid of our patients with SS. One might speculate that the elevated tear fluid IL-6 levels are a consequence of concentration because of the small volume of tears found in patients with SS.
Tishler et al 䡠 Tear IL-6 Levels in SS However, several arguments can be raised against this determination. Although small volumes of tear fluid are the hallmark of SS, concentrations of different tear fluid constituents vary. While concentrations of lactoferrin and lysozyme and peroxidase are reduced, increased concentrations of 2-microglobulin and ␣1 antitrypsine are found in the tear fluid of patients with SS. Furthermore, no correlation between IL-6 tear fluid levels and age, disease duration, or Schirmer’s test could be found in our group of patients as in another study concerning 2-microglobulin levels.13 Interleukin-6, which is a multifactorial cytokine, is involved in T-cell differentiation, stem cell proliferation, and acutephase protein production.16 The concept that quite a simple noninvasive method, which does not require specific equipment, can help in the diagnosis of SS is attractive. To the best of our knowledge, this is the first study that shows elevated IL-6 levels in the tear fluid of patients with SS. Because the rise in IL-6 might be just a marker of CD4⫹ lymphocytic accumulation, further studies with larger numbers of patients, which will include other autoimmune disorders and nonimmune-mediated inflammatory eye diseases, are needed to determine the specificity of this finding for SS.
References 1. Jacobsson LT, Axell TE, Hansen BU, et al. Dry eyes or mouth-an epidemiological study in Swedish adults, with special reference to primary Sjo¨gren’s syndrome. J Autoimmun 1989;2:521–7. 2. Prause JU. Dry Eye in Sjo¨gren’s Syndrome-State of the Art. Homma M, Sugai S, Tojo T, Miyasaka N, Akuzuki M, eds. Amsterdam: Kugler Publications, 1994;5– 8. 3. A comprehensive guide. Lemp MA, Marquardt R, eds. The Dry Eye: Berlin; New York: Springer–Verlag, 1992;65–99. 4. Pepose JS, Akata RF, Pflugfelder SC, Voigt W. Mononuclear cell phenotypes and immunoglobulin gene rearrangements in
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
lacrimal gland biopsies for patients with Sjo¨gren’s syndrome. Ophthalmology 1990;97:1599 – 605. Saito I, Terauchi K, Shimuta M, et al. Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjo¨gren’s syndrome. J Clin Lab Anal 1993;7:180 –7. Virtanen T, Konttinen YT, Honkanen N, et al. Tear fluid plasmin activity of dry eye patients with Sjo¨gren’s syndrome. Acta Ophthalmol Scand 1997;75:137– 41. Fox RI, Kang HI, Ando D, et al. Cytokine mRNA expression in salivary gland biopsies of Sjo¨gren’s syndrome. J Immunol 1994;152:5532–9. Boumba D, Skopouli FN, Moutsopoulos HM. Cytokine mRNA expression in the labial salivary gland tissues from patients with primary Sjo¨gren’s syndrome. Br J Rheumatol 1995;34:326 –33. Vitali C, Bombardieri S, Moutsopoulos HM, et al. Preliminary criteria for the classification of Sjo¨gren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 1993;36:340 –7. Prause JU. Clinical ophthalmological tests for the diagnosis of keratoconjunctivitis sicca. Clin Exp Rheumatol 1989;7: 141– 4. Pflugfelder SC, Tseng SCG, Sanabria O, et al. Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation. Cornea 1998;17:38 –56. Markusse HM, van Haeringen NJ, Swaak AJ, et al. Tear fluid analysis in primary Sjo¨gren’s syndrome. Clin Exp Rheumatol 1993;11:175– 8. Markusse HM, Huysen JC, Nieuwenhuys EJ, Swaak AJ. Beta 2-microglobulin in tear fluid from patients with primary Sjo¨gren’s syndrome. Ann Rheum Dis 1992;51:503–5. Tishler M, Yaron I, Shirazi I, Yaron M. Saliva: an additional diagnostic tool in Sjo¨gren’s syndrome. Semin Arthritis Rheum 1997;27:173–9. Grisius MM, Bermudez DK, Fox PC. Salivary and serum interleukin 6 in primary Sjo¨gren’s syndrome. J Rheumatol 1997;24:1089 –91. Brach MA, Hermann F. Interleukin 6: presence and future. Int J Clin Lab Res 1992;22:143–51.
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