European Journal of Endocrinology (2004) 150 225–234 ISSN 0804-4643 EXPERIMENTAL STUDY The role of CXCR5 and its ligand CXCL13 in the compartmentalization of lymphocytes in thyroids affected by autoimmune thyroid diseases G Aust, D Sittig, L Becherer1, U Anderegg2, A Schu¨tz3, P Lamesch1 and E Schmu¨cking Institute of Anatomy, 1Department of Surgery, 2Department of Dermatology and 3 Institute of Pathology, University of Leipzig, Leipzig, Germany (Correspondence should be addressed to G Aust, University of Leipzig, Institute of Anatomy, Ph-Rosenthal-Strasse 55, Leipzig, 04103, Germany; Email: [email protected]) Abstract Objective: Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are characterized by lymphocytic infiltrates partly resembling secondary lymphoid follicles in the thyroid. CXCR5 and its ligand CXCL13 regulate compartmentalization of B- and T-cells in secondary lymphoid organs. The aim of the study was to elucidate the role of this chemokine receptor–ligand pair in thyroid autoimmunity. Methods: Peripheral blood and thyroid-derived lymphocyte subpopulations were examined by flow cyto- metry for CXCR5. CXCR5 and CXCL13 cDNA were quantified in thyroid tissues by real-time RT-PCR. Results: We found no differences between the percentages of peripheral blood CXCR5þ T- and B-cells in GD patients (n ¼ 10) and healthy controls (n ¼ 10). In GD patients, the number of memory CD4þ cells expressing CXCR5 which are functionally characterized as follicular B helper T-cells is higher in thyroid- derived (18^3%) compared with peripheral blood T-lymphocytes (8^2%). The highest CXCL13 mRNA levels were found in HT (n ¼ 2, 86.1^1.2 zmol (10221 mol) cDNA/PCR) followed by GD tissues (n ¼ 16, 9.6^3.5). Only low amounts were determined in thyroid autonomy (TA) (n ¼ 11) thyroid tissues, irre- spective of whether the autonomous nodule (0.5^0.1) or the surrounding normal tissue (1.8^0.7) had been analyzed. The same differences were found for CXCR5 (HT: 179.1^6.8; GD: 17.4^10.6; TAnodule: 0.8^0.5; TAnormal: 4.4^3.6). In GD, there is a correlation between CXCL13 and CXCR5 mRNA levels and the number of focal lymphocytic infiltrates and germinal centers as well as anti-thyroperoxidase but not anti-TSH receptor autoantibodies. Conclusions: CXCR5 and CXCL13 play an essential role in maintaining B- and T-cells in lymphocytic infil- trates and ectopic follicles in thyroid tissue from patients affected by autoimmunity. European Journal of Endocrinology 150 225–234 Introduction representing follicular dendritic cells. In addition to B- cells, CXCR5 is expressed in a subset of memory þ Lymphoid follicles are usually observed in secondary CD4 T-cells termed follicular B-helper T-cells (TFH) lymphoid tissues and organs, and are essential struc- that migrate into the B-cell zone to support immuno- tures for normal immune responses in which antigen- globulin production (5–8). specific B- and T-cells are expanded against antigens. In addition to lymphoid follicles located in secondary Chemokines and their receptors are involved in com- lymphoid tissues and organs, follicle formation is partmentalization of these follicles establishing spatially observed ectopically in chronic inflammatory sites or and functionally segregated microenvironments. The tissues affected by autoimmunity. Here, they recapitu- role of CXCR5 and its unique ligand CXCL13 (BCA-1; late the structure of normal secondary follicles. Ectopic B-cell attracting chemokine 1) in the organization of lymphoid follicles have been demonstrated in synovium lymphoid follicles is clearly demonstrated in gene- from chronic arthritis patients (9, 10), in the salivary targeted mice (1–3). Interaction between CXCR5 and gland from patients with Sjogren’s syndrome (11), CXCL13 is especially required for B-cell architectural and in intestinal specimens from patients with colitis organization (4). In CXCL13-deficient mice, B-cells ulcerosa (12). CXCL13 and CXCR5 are expressed in failed to organize in polarized follicular clusters and these irregular aggregates, suggesting that both may instead appeared as a ring of cells at the perimeter of play a role in aberrant lymphoid tissue. T-cell areas. In autoimmune thyroid diseases, ectopic lymphoid Naive B-cells localize in a CXCR5-dependent manner follicles have been demonstrated in thyroid tissues from in the B-cell zone of the normal lymphoid follicle patients with Hashimoto’s thyroiditis (HT) as well as where CXCL13 is produced by stromal cells mainly Graves’ disease (GD). Lymphocytic infiltration q 2004 Society of the European Journal of Endocrinology Online version via http://www.eje.org Downloaded from Bioscientifica.com at 09/30/2021 05:59:34AM via free access 226 G Aust and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2004) 150 progressively replaces thyroid follicles in HT. A spectrum and TA (n ¼ 11, five females, 55.7^5.2 years) were of lymphocytic infiltration is observed in GD from the diagnosed on the basis of clinical, biochemical and minimal numbers of immune cells in some thyroids immunological characteristics and from scintiscans (diffuse infiltration) to focal thyroiditis in others (13). (Table 1). Thyroid scans using Tc-99m pertechnetate In contrast to HT, most of the thyroid in GD remains from all patients with unifocal TA showed a single intact except for signs of hyperfunction. In most cases, hot nodule and a low uptake in the rest of the thyroid. there is a direct relationship between the extent of lym- Hyperthyroidism was confirmed by measurements of phocytic infiltration in GD tissues and the percentage of serum thyroid-stimulating hormone (TSH), free thyrox- germinal center (GC) formation. The degree of lympho- ine and free triiodothyronine. The diagnosis of GD was cytic infiltration was related to the titers of anti-thyroglo- also made on a clinical basis, which included the bulin and anti-thyroperoxidase (TPO) antibodies (13). presence of anti-thyroid antibodies, goiter and typical T-cells were mostly scattered individually or in small symptoms and signs of thyrotoxicosis. The diagnosis groups between the follicles; however, they mainly was confirmed by histopathological diagnosis. All GD formed clusters in the severely infiltrated group (14). patients had been treated with anti-thyroid drugs Recent studies suggest that chemokines and their since diagnosis (Table 1). receptors are not only involved in the initiation and Antibodies against TSH receptor (TSH-R) (TSH-bind- maintenance of the autoimmune process in HT and ing-inhibiting immunoglobulin, TBII) and TPO were GD (15–18), but are also capable of organizing and measured in serum obtained up to 2 weeks before oper- sustaining lymphoid follicles in the thyroid (17, 19). ation using commercial RIA kits (TRAKhuman DYNO- Armengol et al. (17) detected CXCL13 mRNA in thyr- TEST, anti-TPOn DYNOTEST; Brahms Diagnostica oids affected by autoimmunity, but not in normal thyr- GmbH, Berlin, Germany). All GD patients showed oid. We published the first quantitative results on positive TBII (.2 IU/l), and 9 out of 16 showed positive CXCL13 mRNA levels in autoimmune and non-auto- anti-TPO antibodies (.60 U/ml). PBLs of GD patients immune thyroid tissues (20), which were very recently were investigated prior to surgery. Ten out of the confirmed by others (19). All studies have shown that sixteen patients were selected for flow cytometry the presence of chemokines in most thyroids is largely studies. GD patients with low lymphocytic infiltration restricted to lymphocytic cell infiltration. However, and low or negative TPO antibody levels are rare. thyrocytes and thyroid-derived fibroblasts are also Thyroid tissues from such patients were taken from able to express some chemokines (21–25), and may our established thyroid tissue bank. Because we thus perturb the immune process. needed freshly isolated PBLs and TLs for flow The aim of our study was to evaluate whether any cytometry analysis, the ten other GD patients were difference in CXCR5 expression between thyroid-derived included in the present study when they underwent lymphocytes (TLs) and peripheral blood lymphocytes surgery. (PBLs) within memory CD4þ and CD8þ T-cell and Peripheral blood samples from ten normal adults B-cell fractions had occurred. Moreover, we clarified (two male, eight female; mean age 33.7^2.7 years) the question of whether intrathyroidal CXCR5 and without any history of autoimmune disease were used CXCL13 mRNA levels were correlated with the presence as controls. The study was approved by the local Com- and extension of focal lymphocytic infiltration in thyr- mittee of Medical Ethics, and all patients gave their oids affected by GD and HT. In contrast to Armengol written consent. et al. (19), who included multinodular goiter tissues as controls, we compared the results with thyroid Real-time PCR tissue from patients with non-autoimmune thyroid autonomy (TA). TA nodules, nearly completely free of Total cellular RNA was isolated using the QIAGEN total any lymphocyte, are a useful background control; RNA isolation kit (QIAGEN GmbH, Hilden, Germany). normal, nodule-surrounding TA tissues mimic the cDNA was synthesized from 1 mg RNA in a 20 ml stan- normal thyroid situation. In addition to Armengol dard reaction mixture containing 200 U Superscript II et al. (19), we also quantified CXCR5 mRNA levels as RNaseH reverse transcriptase (Invitrogen GmbH, an extent of the number of CXCR5þ cells. Karlsruhe, Germany). Our results demonstrated that CXCR5 and its ligand Quantitative PCR was performed on a Rotorgene are indeed involved in the organization and extension (Corbett Research, Mortlake, Australia) real-time of lymphocytic infiltrates and ectopic follicles. machine by using SYBR Green I (Molecular Probes Europe, Leiden, The Netherlands) as a double-strand DNA-specific binding dye and continuous fluorescence Materials and methods monitoring. Amplification was carried out in a total Patients volume of 20ml containing 0.5 mmol/l of each primer, 2.5 mmol/l MgCl2, 1 U Taq Polymerase (Roche), 10 £ GD (n ¼ 16, all females, mean age^S.E.M.