Clin. exp. Immunol. (1992) 88, 405-409
Soluble IL-2 receptor levels in patients with Graves' ophthalmopathy M. F. PRUMMEL*, W. M. WIERSINGA*, R. VAN DER GAAGt, M. P. MOURITSt & L. KOORNNEEFt *Department of Endocrinology and tOrbital Centre, Academic Medical Centre, University of Amsterdam, Amsterdam, and TDepartment of Ophthalmo-Immunology, Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands
(Acceptedfor publication 26 February 1992)
SUMMARY In various autoimmune diseases circulating levels of soluble IL-2 receptor (sIL-2R) seem to be related to disease activity. Because reliable parameters of disease activity in Graves' ophthalmopathy are lacking, we measured sIL-2R levels in 47 patients with this disorder. The patients had Graves' disease, but no other immune-mediated diseases, had not yet received specific treatment for their ophthalmopathy and were euthyroid during the entire study period. Twenty-one of the 47 patients (45%) had sIL-2R values above the upper normal limit of 650 U/ml, as established in 20 healthy controls. There were no differences between patients with normal (median 469, range 280-644 U/ml) and elevated (median 946, range 678-1588 U/ml) sIL-2R levels regarding duration or severity of the eye disease (as assessed clinically from the total eye score). However, patients with severely enlarged eye muscles had higher sIL-2R values than patients with less severely enlarged eye muscles on CT scan. Patients with elevated sIL-2R tended to have a higher response rate (71 %) to a 3-month course of prednisone, than those with normal levels (46%; P=0-081). Since a successful outcome of prednisone treatment might be representative for disease activity, the elevated sIL-2R levels seem to reflect active inflammation. Although the practical relevance of this finding in individual patients is limited, it underscores the importance of cell-mediated immune responses in this thyroid-related eye disease. Keywords IL-2 receptor Graves' ophthalmopathy prednisone thyroid diseases
INTRODUCTION IL-2 plays a pivotal role in the immune response and various cells have membrane-bound receptors for this cytokine [1]. Resting lymphocytes express an IL-2 receptor (IL-2R) of intermediate affinity, while expression of a second protein (Tac molecule) with low affinity is induced after activation [2]. When both proteins are linked together, a high-affinity receptor for IL2 is formed. Activated lymphocytes also release a truncated form of the Tac molecule with a low affinity for IL-2 [2]. This soluble IL-2R (sIL-2R) can be measured with an ELISA [3]. Since the introduction of a commercially available ELISA, sIL-2R has been measured in many immune-mediated diseases [1], and circulating levels appear to correlate with disease activity in systemic lupus erythematosus (SLE) [4,5] and rheumatoid arthritis [6-8]. Two studies found elevated sIL-2R levels in untreated patients with Graves' hyperthyroidism, returning to normal after anti-thyroid treatment [9,10]. In addition, it has been shown that thyrotoxicosis can result in increased sIL-2R levels [11]. Correspondence: Mark F. Prummel, MD, Department of Endocrinology, F5-258, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
405
Graves' ophthalmopathy is a disease closely linked to Graves' thyroid disease (GTD) [12]. It is considered to be an organ-specific autoimmune disease, but it is uncertain whether cellular or humoral immunological mechanisms are primarily disturbed [13]. Corticosteroids are the mainstay of immunosuppressive treatment, to which about 65% of patients respond [14,15]. The factors determining the response are unknown, but it is conceivable that a favourable response is associated with the activity of the autoimmune process [16]. To spare patients with inactive eye disease the side-effects of prednisone, reliable parameters of disease activity are necessary [17], but as yet unavailable. Therefore, we studied whether measuring sIL-2R levels could be helpful in delineating the activity of this disease. PATIENTS AND METHODS Patients Sixty-four consecutive patients with Graves' ophthalmopathy, who participated in prospective studies evaluating the effect of prednisone on their eye status, were included. All patients had the typical clinical features of Graves' ophthalmopathy and were euthyroid for at least 2 months before start of treatment (thyroxine (T4), 75-150 nmol/l, tri-iodothyronine (T3), 1-~302~45 nmol/l, and thyroid-stimulating hormone (TSH), .45
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M. F. Prummel et al. Table 1. Comparison of clinical data in 47 patients with Graves' ophthalmopathy with normal (650 U/ml) soluble IL-2 receptor (sIL-2R) levels prior to treatment
Number (%) sIL-2R Age (years) Sex: F/M T4 (nmol/l) T3 (nmol/l) TSH (mU/l) Duration Graves' ophthalmopathy (months) Duration GTD (months) MsAb -/±/+/++ TgAb-//+/+ + Methimazole No methimazole Severity of Eye Disease: Total Eye Score NOSPECS class 2 0/a/b/c 3 proptosis (mm) 4 0/a/b 6 0/a/b Eye muscle score (EMS) EMS 1-3 EMS 4-5
sIL-2R 650
26 (55%) 469 (280-644) 47-1 + 11 9 21/5 123+22 1-94+0 24 0-8+1-0 14 (5-120) 25 (3-300) 12/7/3/4 16/5/1/4 10 16
21 (45%) 946 (678-1588) 49 7+ 13 6 17/4 122+35 1 90+0-29 0 7+ 1-3 12 (6-96) 20 (5-96) 7/6/3/5 14/1/2/4 12 9
10-9+3 4 Nr of patients 2/13/10/1 20-8 + 3-3 1/8/17 25/1/0 1-9+ 1-5 n = 22 (96%) n= 1 (4%)
12 8+7-3
NS
4/11/3/3 21-1 +±31 0/5/16 19/1/1 2-5 + 1 9 n = 14 (74%) n = 5 (26%)
NS NS NS NS 0-04
P 650 U/ml: 71% versus 46°/.; P=0*081 by x2 test. Sensitivity, 56%; specificity, 70%; positive predictive value, 71%; negative predictive value, 54%.
- '_ Controls (n=20) Responders ( n=27) Non-responders (n=20) Fig. 1. Pretreatment levels of soluble IL-2 receptor (sIL-2R) in healthy controls, and in responders and non-responders to a 12-week course of prednisone. The median values in the different groups are indicated.
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sIL-2R in Graves' ophthalmopathy changes, multiplied by the grade within that class (a =1; b = 2; and c=3) [15], and the eye muscle score (EMS), as assessed blindly from pretreatment coronal CT scans, and scored on a scale from 1 (minimally enlarged) to 5 (extremely enlarged eye muscles) [15]. Finally, we assessed the predictive value of sIL-2R for the therapeutic outcome of prednisone treatment. It was defined as response (decrease in NOSPECS class, or, if this did not occur, in grade within a class; class 5 was excluded from this analysis), or as non-response (no change; or failure, defined as an increase in NOSPECS class or grade). If both increases and decreases occurred, the change in the highest class defined the therapeutic outcome. sIL-2R was measured using a commercially available ELISA kit (T Cell Sciences, Cambridge, MA) and expressed in U/ml. When 17 samples with varying sIL-2R values were analysed at a 1: 5 dilution, 99 + 20% (mean + s.d.; range 63134%) of the values of the undiluted samples were obtained. T4 and T3 were measured by in-house radioimmunoassays (RIA); TSH was determined either by RIA or by immunoradiometric assay. Anti-microsomal (MsAb) and anti-thyroglobulin (TgAb) autoantibodies were measured semi-quantitatively by a conventional immunofluorescence technique. Data are expressed as mean + s.d., or as median (range). Differences in sIL-2R between groups were analysed by the Mann-Whitney U-tests, and changes in sIL-2R after prednisone by the Wilcoxon rank sum test. For data with a normal distribution Student's t-tests were used. x2 tests were applied to compare percentages between groups. RESULTS No differences were observed in age and sex between the 47
Graves' ophthalmopathy patients (19% men; age 48-2+12-6 years) and the 20 controls (15% men; age 43-5 + 128 years, NS). Patients had higher sIL-2R levels (median 635, range 280-1588 U/ml) than controls (385, range 215-633 U/ml; P 650 U/ml) or with normal (< 650 U/ml) sIL-2R levels (Table 1). Ten patients had Graves' ophthalmopathy without a history of thyroid disease, and four of these had elevated sIL-2R values. The other 36 patients had been or still were treated for GTD with anti-thyroid drugs (mostly methimazole in combination with thyroxine). The number of patients using these drugs and the mean dosage was similar in patients with normal or elevated sIL-2R levels, as were the levels of MsAb and TgAb (Table 1). As for ophthalmopathy, patients with elevated and those with normal sIL-2R levels had a similar duration and severity of Graves' ophthalmopathy, but patients with elevated sIL-2R levels had a higher EMS than those with normal values (Table 1). Also, patients with severely enlarged eye muscles (EMS 4-5; n =6) had higher sIL-2R levels than did those with a lesser degree of enlargement (EMS 1-3; n = 36): median 869 (6281588) versus 629 (280-1364) U/ml (P= 0-018). Patients with sIL-2R > 650 U/ml tended to have a higher response rate to prednisone treatment than did those with normal levels, but the predictive value of sIL-2R for the therapeutic outcome was rather low (Table 2). Also, there were no differences in pretreatment sIL-2R values between those who responded to corticosteroid treatment and those who did not (Fig. 1).
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Fig. 2. The effect of a 12-week course of oral prednisone on soluble IL-2 receptor (sIL-2R) levels in 26 patients with Graves' ophthalmopathy, and in responders and non-responders to this therapy (excluding two patients with a relapse). to, before start of treatment. t12, after 12 weeks of prednisone. The median values in the different groups are indicated.
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Prednisone treatment was associated with a decrease in sIL2R levels (Fig. 2). In three patients a marked increase after 12 weeks of prednisone was seen: one had an exceptionally favourable response to treatment, and in two this rise in sIL-2R levels preceded a clinical relapse shortly after discontinuation of prednisone. Excluding the latter two patients, a significant decrease in sIL-2R levels was seen in responders and nonresponders, although a greater fall was found in the 14 responders (median 323 U/ml) than in the 10 non-responders (142 U/ml; P=0 053).
DISCUSSION We found elevated sIL-2R levels in 45% of euthyroid patients with moderate to severe Graves' ophthalmopathy. This could not be explained by the levels of antithyroid autoantibodies or the duration of GTD. Because we excluded patients with other diseases associated with immunological disturbances, it is likely that the ophthalmopathy itself was responsible for the elevated sIL-2R levels. This is in accordance with a recent study in which higher sIL-2R levels were found in infiltrative eye disease, than in patients with Graves' hyperthyroidism without eye changes [18]. It is not clear why 45% of the patients had elevated sIL-2R levels. There was no correlation with the severity of the ophthalmopathy (using total eye score and NOSPECS), but higher values were found in a subgroup with severely enlarged eye muscles on CT scan. Since serum sIL-2R is considered to be derived from local, e.g. intra-orbital, activated lymphocytes, it might be that serum levels depict the active inflammatory process reliably only when a considerable amount of tissue is involved. In patients with rheumatoid arthritis and SLE, serum sIL2R levels appear to reflect disease activity [4-8] and therefore it is conceivable that sIL-2R levels in Graves' ophthalmopathy primarily reflect the activity, rather than the severity of the disease. However, there is no validated method to assess disease activity, but it is likely that patients with active eye disease respond better to corticosteroids than do those with inactive, fibrotic disease [19]. Using this approach, we found that patients with elevated sIL-2R tended to have a higher response rate to prednisone than did those with normal levels. Together with the relation between sIL-2R and eye muscle enlargement, which is probably caused by marked lymphocytic infiltration and oedema, this might indicate that sIL-2R levels correspond with the activity of the autoimmune process. However, the predictive value of sIL-2R for treatment outcome was rather poor. Measuring sIL-2R alone is therefore not helpful in individual patients, but might serve as a useful adjunct to a clinical activity score. It is still controversial whether Graves' ophthalmopathy is caused by a dysfunction of cellular or humoral immunity [20,21]. Although sIL-2R can be released by activated T as well as B cells [2], its predominant source is the activated T lymphocyte [22]. Therefore, our findings indicate that cellular immunity plays a role in Graves' ophthalmopathy, as was recently stressed by Weetman [13]. This is in agreement with the detection of a monocyte migration inhibitory factor in patients with active eye disease [23]. In a recent study no abnormal sIL2R production was found after mitogen stimulation of peripheral blood lymphocytes from patients with ophthalmopathy,
which might be due to the fact that circulating T cells may not reflect the activity of the intra-orbital cell population [24]. The fall of sIL-2R levels upon treatment with prednisone, which seemed to be greater in responders than in nonresponders, might result from inactivation of the immune attack; alternatively, it could reflect a less specific change in IL-2 production and a subsequent decrease in IL-2R expression [25]. Interestingly, in two patients a relapse was preceded by a marked rise in sIL-2R levels, as in rheumatoid arthritis [26] and in Graves' hyperthyroidism, where a rise in sIL-2R heralded a relapse [18]. We found elevated sIL-2R levels in 45% of untreated euthyroid patients with Graves' ophthalmopathy, which seemed to correlate with the degree of eye muscle enlargement and with disease activity. Patients with elevated sIL-2R levels tended to have a greater probability to respond to prednisone, though the clinical relevance in individual patients is limited. Our results underscore the importance of cell-mediated immunity in the pathogenesis of Graves' ophthalmopathy.
ACKNOWLEDGMENTS Presented in part at the International Symposium on Graves' Ophthalmopathy, Amsterdam, The Netherlands, 23-24 August, 1991, and at the 19th annual meeting of the European Thyroid Association, Hannover, Germany, 25-30 August, 1991. We are grateful for the technical assistance of Mrs Frederiek de Wilde of the Laboratory of Clinical Immunology.
REFERENCES I Rubin LA, Nelson DL. The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Intern Med 1990; 113:61927. 2 Rubin LA. The soluble interluekin-2 receptor in rheumatic disease. Arthritis Rheum 1990; 33:1145-8. 3 Rubin LA, Kurman CC, Fritz ME, Biddison WE, Boutin B, Yarchoan R, Nelson DL. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro. J Immunol 1985; 135:3172-7. 4 Tokano Y, Murashima A, Takasaki Y, Hashimoto H, Okumura K, Hirose S. Relation between soluble interleukin 2 receptor and clinical findings in patients with systemic lupus erythematosus. Ann Rheum Dis 1989; 48:803-9. 5 Campen DH, Horwitz DA, Quismorio FP, Ehresmann GR, Martin WJ. Serum levels of interleukin-2 receptor and activity of rheumatic diseases characterized by immune system activation. Arthritis Rheum 1988; 31:1358-64. 6 Rubin LA, Snow KM, Kurman CC, Nelson DL, Keystone EC. Serial levels of soluble interluekin 2 receptor in the peripheral blood of patients with rheumatoid arthritis: correlations with disease activity. J Rheumatol 1990; 17:597-602. 7 Symons JA, Wood NC, Di Giovine FS, Duff GW. Soluble IL-2 receptor in rheumatoid arthritis. Correlation with disease activity, IL-1 and IL-2 inhibition. J Immunol 1988; 141:2612-18. 8 Semenzato G, Bambara LM, Biasi D, et al. Increased serum levels of soluble interleukin-2 receptor in patients with systemic lupus erythematosus and rheumatoid arthritis. J Clin Immunol 1988; 8:447-52. 9 Chow CC, Lai KN, Leung JCK, Chan JCN, Cockram CS. Serum soluble interleukin 2 receptor in hyperthyroid Graves' disease and effect of carbimazole therapy. Clin Endocrinol 1990; 33:317-21. 10 Wilson R, McKillop JH, Buchanan LM, Bradley H, Smith WE, Thomson JA. The effect of carbimazole therapy on interleukin 2, interleukin 2 receptors and free radicals. Autoimmunity 1990; 8:3-7.
sIL-2R in Graves' ophthalmopathy 11 Koukkou E, Panayiotidis P. Alevizou-Terzaki V, Thalassinos N. High levels of serum soluble interleukin-2 receptors in hyperthyroid patients: correlation with serum thyroid hormones and independence from the etiology of the hyperthyroidism. J Clin Endocrinol Metab 1991; 73:771-6. 12 Jacobson DH, Gorman CA. Endocrine opthalmopathy: current ideas concerning etiology, pathogenesis, and treatment. Endocrin Rev 1984; 5:200-20. 13 Weetman AP. Thyroid-associated eye disease: pathophysiology. Lancet 1991; 338:25-8. 14 Wiersinga WM, Smit T, Schuster-Uittenhoeve AL, van der Gaag R, Koornneef L. Therapeutic outcome of prednisone medication and of orbital irradiation in patients with Graves' ophthalmopathy. Ophthalmologica 1988; 197:75-84. 15 Prummel MF, Mourits MPH, Berghout A, et al. Prednisone and cyclosporin in the treatment of severe Graves' ophthalmopathy. N Engl J Med 1989; 321:1353-9. 16 Fells P. Management of dysthyroid eye disease. Br J Ophthalmol 1991; 75:245-6. 17 Bahn RS, Garrity JA, Gorman CA. Clinical review 13. Diagnosis and management of Graves' ophthalmopathy. J Clin Endocrinol Metab 1990; 71:559-63. 18 Balazs C, Farid NR. Soluble interleukin-2 receptor in sera of patients with Graves' disease. J Autoimmunity 1991; 4:681-8. 19 Mourits MP, Koornneef L, Wiersinga WM, Prummel MF, Berg-
20 21 22
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25
26
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hout A, vd Gaag R. Clinical criteria for the assessment of disease activity in Graves' ophthalmopathy: a novel approach. Br J Opthalmol 1989; 73:639-44. Perros P, Kendall-Taylor P. The pathogenesis of thyroid-associated ophthalmopathy. J Endocrinol 1989; 122:619-24. Jones IR. The pathogenesis and treatment of Graves' ophthalmopathy. Postgrad Med J 1987; 63:731-3. Nelson DL, Rubin LA, Kurman CC, Fritz ME, Boutin B. An analysis of the cellular requirements for the production of soluble interleukin-2 receptors in vitro. J Clin Immunol 1986; 6:114-20. Van der Gaag R, Broersma L, Mourits MP, Koornneef L, Wiersinga WM, Prummel MF, Berghout A. Circulating monocyte inhibitory factor in serum of Graves' ophthalmopathy patients: a parameter for disease activity? Clin Exp Immunol 1989; 75:275-9. Lai KN, Leung JCK, Chow CC, Cockram CS. T lymphocyte activation in euthyroid Graves' ophthalmopathy: soluble interleukin 2 receptor release, cellular interleukin 2 receptor expression and interleukin 2 production. Acta Endocrinol 1989; 120:602-9. Boumpas DT, Anastassiou ED, Older SA, Tsokos GC, Nelson DL, Balow JE. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription. J Clin Invest 1991; 87:1739-47. Wood NC, Symons JA, Duff GW. Serum Interleukin-2-receptor in rheumatoid arthritis: a prognostic indicator of disease activity? J Autoimmunity 1988; 1:353-61.
Soluble IL-2 receptor levels in patients with Graves' ophthalmopathy M. F. PRUMMEL*, W. M. WIERSINGA*, R. VAN DER GAAGt, M. P. MOURITSt & L. KOORNNEEFt *Department of Endocrinology and tOrbital Centre, Academic Medical Centre, University of Amsterdam, Amsterdam, and TDepartment of Ophthalmo-Immunology, Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands
(Acceptedfor publication 26 February 1992)
SUMMARY In various autoimmune diseases circulating levels of soluble IL-2 receptor (sIL-2R) seem to be related to disease activity. Because reliable parameters of disease activity in Graves' ophthalmopathy are lacking, we measured sIL-2R levels in 47 patients with this disorder. The patients had Graves' disease, but no other immune-mediated diseases, had not yet received specific treatment for their ophthalmopathy and were euthyroid during the entire study period. Twenty-one of the 47 patients (45%) had sIL-2R values above the upper normal limit of 650 U/ml, as established in 20 healthy controls. There were no differences between patients with normal (median 469, range 280-644 U/ml) and elevated (median 946, range 678-1588 U/ml) sIL-2R levels regarding duration or severity of the eye disease (as assessed clinically from the total eye score). However, patients with severely enlarged eye muscles had higher sIL-2R values than patients with less severely enlarged eye muscles on CT scan. Patients with elevated sIL-2R tended to have a higher response rate (71 %) to a 3-month course of prednisone, than those with normal levels (46%; P=0-081). Since a successful outcome of prednisone treatment might be representative for disease activity, the elevated sIL-2R levels seem to reflect active inflammation. Although the practical relevance of this finding in individual patients is limited, it underscores the importance of cell-mediated immune responses in this thyroid-related eye disease. Keywords IL-2 receptor Graves' ophthalmopathy prednisone thyroid diseases
INTRODUCTION IL-2 plays a pivotal role in the immune response and various cells have membrane-bound receptors for this cytokine [1]. Resting lymphocytes express an IL-2 receptor (IL-2R) of intermediate affinity, while expression of a second protein (Tac molecule) with low affinity is induced after activation [2]. When both proteins are linked together, a high-affinity receptor for IL2 is formed. Activated lymphocytes also release a truncated form of the Tac molecule with a low affinity for IL-2 [2]. This soluble IL-2R (sIL-2R) can be measured with an ELISA [3]. Since the introduction of a commercially available ELISA, sIL-2R has been measured in many immune-mediated diseases [1], and circulating levels appear to correlate with disease activity in systemic lupus erythematosus (SLE) [4,5] and rheumatoid arthritis [6-8]. Two studies found elevated sIL-2R levels in untreated patients with Graves' hyperthyroidism, returning to normal after anti-thyroid treatment [9,10]. In addition, it has been shown that thyrotoxicosis can result in increased sIL-2R levels [11]. Correspondence: Mark F. Prummel, MD, Department of Endocrinology, F5-258, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
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Graves' ophthalmopathy is a disease closely linked to Graves' thyroid disease (GTD) [12]. It is considered to be an organ-specific autoimmune disease, but it is uncertain whether cellular or humoral immunological mechanisms are primarily disturbed [13]. Corticosteroids are the mainstay of immunosuppressive treatment, to which about 65% of patients respond [14,15]. The factors determining the response are unknown, but it is conceivable that a favourable response is associated with the activity of the autoimmune process [16]. To spare patients with inactive eye disease the side-effects of prednisone, reliable parameters of disease activity are necessary [17], but as yet unavailable. Therefore, we studied whether measuring sIL-2R levels could be helpful in delineating the activity of this disease. PATIENTS AND METHODS Patients Sixty-four consecutive patients with Graves' ophthalmopathy, who participated in prospective studies evaluating the effect of prednisone on their eye status, were included. All patients had the typical clinical features of Graves' ophthalmopathy and were euthyroid for at least 2 months before start of treatment (thyroxine (T4), 75-150 nmol/l, tri-iodothyronine (T3), 1-~302~45 nmol/l, and thyroid-stimulating hormone (TSH), .45
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M. F. Prummel et al. Table 1. Comparison of clinical data in 47 patients with Graves' ophthalmopathy with normal (650 U/ml) soluble IL-2 receptor (sIL-2R) levels prior to treatment
Number (%) sIL-2R Age (years) Sex: F/M T4 (nmol/l) T3 (nmol/l) TSH (mU/l) Duration Graves' ophthalmopathy (months) Duration GTD (months) MsAb -/±/+/++ TgAb-//+/+ + Methimazole No methimazole Severity of Eye Disease: Total Eye Score NOSPECS class 2 0/a/b/c 3 proptosis (mm) 4 0/a/b 6 0/a/b Eye muscle score (EMS) EMS 1-3 EMS 4-5
sIL-2R 650
26 (55%) 469 (280-644) 47-1 + 11 9 21/5 123+22 1-94+0 24 0-8+1-0 14 (5-120) 25 (3-300) 12/7/3/4 16/5/1/4 10 16
21 (45%) 946 (678-1588) 49 7+ 13 6 17/4 122+35 1 90+0-29 0 7+ 1-3 12 (6-96) 20 (5-96) 7/6/3/5 14/1/2/4 12 9
10-9+3 4 Nr of patients 2/13/10/1 20-8 + 3-3 1/8/17 25/1/0 1-9+ 1-5 n = 22 (96%) n= 1 (4%)
12 8+7-3
NS
4/11/3/3 21-1 +±31 0/5/16 19/1/1 2-5 + 1 9 n = 14 (74%) n = 5 (26%)
NS NS NS NS 0-04
P 650 U/ml: 71% versus 46°/.; P=0*081 by x2 test. Sensitivity, 56%; specificity, 70%; positive predictive value, 71%; negative predictive value, 54%.
- '_ Controls (n=20) Responders ( n=27) Non-responders (n=20) Fig. 1. Pretreatment levels of soluble IL-2 receptor (sIL-2R) in healthy controls, and in responders and non-responders to a 12-week course of prednisone. The median values in the different groups are indicated.
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sIL-2R in Graves' ophthalmopathy changes, multiplied by the grade within that class (a =1; b = 2; and c=3) [15], and the eye muscle score (EMS), as assessed blindly from pretreatment coronal CT scans, and scored on a scale from 1 (minimally enlarged) to 5 (extremely enlarged eye muscles) [15]. Finally, we assessed the predictive value of sIL-2R for the therapeutic outcome of prednisone treatment. It was defined as response (decrease in NOSPECS class, or, if this did not occur, in grade within a class; class 5 was excluded from this analysis), or as non-response (no change; or failure, defined as an increase in NOSPECS class or grade). If both increases and decreases occurred, the change in the highest class defined the therapeutic outcome. sIL-2R was measured using a commercially available ELISA kit (T Cell Sciences, Cambridge, MA) and expressed in U/ml. When 17 samples with varying sIL-2R values were analysed at a 1: 5 dilution, 99 + 20% (mean + s.d.; range 63134%) of the values of the undiluted samples were obtained. T4 and T3 were measured by in-house radioimmunoassays (RIA); TSH was determined either by RIA or by immunoradiometric assay. Anti-microsomal (MsAb) and anti-thyroglobulin (TgAb) autoantibodies were measured semi-quantitatively by a conventional immunofluorescence technique. Data are expressed as mean + s.d., or as median (range). Differences in sIL-2R between groups were analysed by the Mann-Whitney U-tests, and changes in sIL-2R after prednisone by the Wilcoxon rank sum test. For data with a normal distribution Student's t-tests were used. x2 tests were applied to compare percentages between groups. RESULTS No differences were observed in age and sex between the 47
Graves' ophthalmopathy patients (19% men; age 48-2+12-6 years) and the 20 controls (15% men; age 43-5 + 128 years, NS). Patients had higher sIL-2R levels (median 635, range 280-1588 U/ml) than controls (385, range 215-633 U/ml; P 650 U/ml) or with normal (< 650 U/ml) sIL-2R levels (Table 1). Ten patients had Graves' ophthalmopathy without a history of thyroid disease, and four of these had elevated sIL-2R values. The other 36 patients had been or still were treated for GTD with anti-thyroid drugs (mostly methimazole in combination with thyroxine). The number of patients using these drugs and the mean dosage was similar in patients with normal or elevated sIL-2R levels, as were the levels of MsAb and TgAb (Table 1). As for ophthalmopathy, patients with elevated and those with normal sIL-2R levels had a similar duration and severity of Graves' ophthalmopathy, but patients with elevated sIL-2R levels had a higher EMS than those with normal values (Table 1). Also, patients with severely enlarged eye muscles (EMS 4-5; n =6) had higher sIL-2R levels than did those with a lesser degree of enlargement (EMS 1-3; n = 36): median 869 (6281588) versus 629 (280-1364) U/ml (P= 0-018). Patients with sIL-2R > 650 U/ml tended to have a higher response rate to prednisone treatment than did those with normal levels, but the predictive value of sIL-2R for the therapeutic outcome was rather low (Table 2). Also, there were no differences in pretreatment sIL-2R values between those who responded to corticosteroid treatment and those who did not (Fig. 1).
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Fig. 2. The effect of a 12-week course of oral prednisone on soluble IL-2 receptor (sIL-2R) levels in 26 patients with Graves' ophthalmopathy, and in responders and non-responders to this therapy (excluding two patients with a relapse). to, before start of treatment. t12, after 12 weeks of prednisone. The median values in the different groups are indicated.
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Prednisone treatment was associated with a decrease in sIL2R levels (Fig. 2). In three patients a marked increase after 12 weeks of prednisone was seen: one had an exceptionally favourable response to treatment, and in two this rise in sIL-2R levels preceded a clinical relapse shortly after discontinuation of prednisone. Excluding the latter two patients, a significant decrease in sIL-2R levels was seen in responders and nonresponders, although a greater fall was found in the 14 responders (median 323 U/ml) than in the 10 non-responders (142 U/ml; P=0 053).
DISCUSSION We found elevated sIL-2R levels in 45% of euthyroid patients with moderate to severe Graves' ophthalmopathy. This could not be explained by the levels of antithyroid autoantibodies or the duration of GTD. Because we excluded patients with other diseases associated with immunological disturbances, it is likely that the ophthalmopathy itself was responsible for the elevated sIL-2R levels. This is in accordance with a recent study in which higher sIL-2R levels were found in infiltrative eye disease, than in patients with Graves' hyperthyroidism without eye changes [18]. It is not clear why 45% of the patients had elevated sIL-2R levels. There was no correlation with the severity of the ophthalmopathy (using total eye score and NOSPECS), but higher values were found in a subgroup with severely enlarged eye muscles on CT scan. Since serum sIL-2R is considered to be derived from local, e.g. intra-orbital, activated lymphocytes, it might be that serum levels depict the active inflammatory process reliably only when a considerable amount of tissue is involved. In patients with rheumatoid arthritis and SLE, serum sIL2R levels appear to reflect disease activity [4-8] and therefore it is conceivable that sIL-2R levels in Graves' ophthalmopathy primarily reflect the activity, rather than the severity of the disease. However, there is no validated method to assess disease activity, but it is likely that patients with active eye disease respond better to corticosteroids than do those with inactive, fibrotic disease [19]. Using this approach, we found that patients with elevated sIL-2R tended to have a higher response rate to prednisone than did those with normal levels. Together with the relation between sIL-2R and eye muscle enlargement, which is probably caused by marked lymphocytic infiltration and oedema, this might indicate that sIL-2R levels correspond with the activity of the autoimmune process. However, the predictive value of sIL-2R for treatment outcome was rather poor. Measuring sIL-2R alone is therefore not helpful in individual patients, but might serve as a useful adjunct to a clinical activity score. It is still controversial whether Graves' ophthalmopathy is caused by a dysfunction of cellular or humoral immunity [20,21]. Although sIL-2R can be released by activated T as well as B cells [2], its predominant source is the activated T lymphocyte [22]. Therefore, our findings indicate that cellular immunity plays a role in Graves' ophthalmopathy, as was recently stressed by Weetman [13]. This is in agreement with the detection of a monocyte migration inhibitory factor in patients with active eye disease [23]. In a recent study no abnormal sIL2R production was found after mitogen stimulation of peripheral blood lymphocytes from patients with ophthalmopathy,
which might be due to the fact that circulating T cells may not reflect the activity of the intra-orbital cell population [24]. The fall of sIL-2R levels upon treatment with prednisone, which seemed to be greater in responders than in nonresponders, might result from inactivation of the immune attack; alternatively, it could reflect a less specific change in IL-2 production and a subsequent decrease in IL-2R expression [25]. Interestingly, in two patients a relapse was preceded by a marked rise in sIL-2R levels, as in rheumatoid arthritis [26] and in Graves' hyperthyroidism, where a rise in sIL-2R heralded a relapse [18]. We found elevated sIL-2R levels in 45% of untreated euthyroid patients with Graves' ophthalmopathy, which seemed to correlate with the degree of eye muscle enlargement and with disease activity. Patients with elevated sIL-2R levels tended to have a greater probability to respond to prednisone, though the clinical relevance in individual patients is limited. Our results underscore the importance of cell-mediated immunity in the pathogenesis of Graves' ophthalmopathy.
ACKNOWLEDGMENTS Presented in part at the International Symposium on Graves' Ophthalmopathy, Amsterdam, The Netherlands, 23-24 August, 1991, and at the 19th annual meeting of the European Thyroid Association, Hannover, Germany, 25-30 August, 1991. We are grateful for the technical assistance of Mrs Frederiek de Wilde of the Laboratory of Clinical Immunology.
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