CXCR6 Within T-Helper (Th) and T-Cytotoxic

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European Journal of Endocrinology (2005) 152 635–643 ISSN 0804-4643

EXPERIMENTAL STUDY CXCR6 within T-helper (Th) and T-cytotoxic (Tc) type 1 lymphocytes in Graves’ disease (GD) G Aust, M Kamprad1, P Lamesch2 and E Schmu¨cking Institute of Anatomy, 1Department of Clinical Immunology and Transfusion Medicine and 2Department of Surgery, University of Leipzig, Phillipp-Rosenthal-Str. 55, Leipzig, 04103, Germany (Correspondence should be addressed to G Aust; Email: [email protected])

Abstract Objective: In Graves’ disease (GD), stimulating anti-TSH receptor antibodies are responsible for hyperthyroidism. T-helper 2 (Th2) cells were expected to be involved in the underlying immune mech- anism, although this is still controversial. The aim of this study was to examine the expression of CXCR6, a chemokine receptor that marks functionally specialized T-cells within the Th1 and T-cytotoxic 1 (Tc1) cell pool, to gain new insights into the running immune processes. Methods: CXCR6 expression was examined on peripheral blood lymphocytes (PBLs) and thyroid- derived lymphocytes (TLs) of GD patients in flow cytometry. CXCR6 cDNA was quantified in thyroid tissues affected by GD (n ¼ 16), Hashimoto’s thyroiditis (HT; n ¼ 2) and thyroid autonomy (TA; n ¼ 11) using real-time reverse transcriptase PCR. Results: The percentages of peripheral CXCR6þ PBLs did not differ between GD and normal subjects. CXCR6 was expressed by small subsets of circulating T-cells and natural killer (NK) cells. CXCR6þ cells were enriched in thyroid-derived T-cells compared with peripheral CD4þ and CD8þ T-cells in GD. The increase was evident within the Th1 (CD4þ interferon-gþ (IFN-gþ)) and Tc1 (CD8þIFN- gþ) subpopulation and CD8þ granzyme Aþ T-cells (cytotoxic effector type). Thyroid-derived fibroblasts and thyrocytes were CXCR62. There was no significant difference between the CXCR6 mRNA levels in GD compared with HT and normal TA tissues. The lowest CXCR6 mRNA levels were obtained from thyroid nodules from TA patients and GD patients with low thyroid peroxidase autoantibody levels. Conclusions: CXCR6 was overexpressed in Th1 and Tc1 TLs compared with PBLs in GD. CXCR6 could be a marker for lymphocytes that have migrated into the thyroid and assist in the thyroid, independently of the bias of the underlying disease.

European Journal of Endocrinology 152 635–643

Introduction type 2 cytokines, respectively. In addition to cytokine secretion, Tc1 and Tc2 populations mediate cytolytic Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) effects, with Tc1 cells utilizing both perforin- and Fas- are organ-specific autoimmune diseases characterized based killing pathways, whereas Tc2 cells primarily by diffuse or focal lymphocytic infiltration. In GD, stim- utilize perforin-mediated cytolysis (1). ulating anti-thyroid-stimulating hormone (TSH) recep- However, recent results from clinical studies and tor (TSHR) autoantibodies overstimulating the TSHR newly established mouse models (2–7) have clearly are responsible for hyperthyroidism. In HT, normal shown that the assumption that GD and HT are thyroid tissue is replaced mainly by lymphocytes orga- immune responses dominated by Th2 or Th1/Tc1, nized in ectopic follicles. The destruction of thyrocytes respectively, is an oversimplification. The role of the causes hypothyroidism. Th1/Th2 balance is especially controversial in murine The expected underlying immune mechanisms of models of Graves’ hyperthyroidism. Suppression of both diseases led to the application of the common TSHR-specific Th1 immune responses by exogenous CD4þ T-helper 1 (Th1)/Th2 paradigm of immunology. IL-4 was associated with inhibition of hyperthyroidism Th2 cells producing interleukin 4 (IL-4), IL-5, IL-10 in BALB/c mice injected with adenovirus-expressing and IL-13 are involved in humoral immunity, as was TSHR (TSHR-adeno model), indicating the critical role first expected for GD. In HT, Th1 cells are thought to of Th1 cytokines (4). In contrast, BALB/c IL-4-knockout produce interferon-g (IFN-g) and act on cell-mediated but not IFN-g-knockout mice failed to develop Graves’ immune responses leading to thyrocyte destruction. hyperthyroidism when injected with TSHR-expressing Similarly to CD4þ T-cells, the CD8þ type 1 cytotoxic M12 B lymphoma cells, suggesting the importance of (Tc1) and Tc2 cells differentially secrete type 1 and endogenous IL-4. In the IL-4- and IFN-g-knockout

q 2005 Society of the European Journal of Endocrinology DOI: 10.1530/eje.1.01892 Online version via www.eje-online.org

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TSHR-adeno model, both IL-4 and IFN-g are implicated blood lymphocytes (PBLs) from the same patients. in the development of hyperthyroidism (7). The potential thyroid-cell sources of the receptor and Besides determining the ratio of type 1 and type 2 its ligand were defined. specific cytokine responses – in most cases IFN-g/IL- 4 – or the immunoglobulin subclasses of TSHR anti- Materials and methods bodies (IgG2a/IgG1), analysis of chemokine receptors and their ligands allows insights into the running auto- Patients immune process (2, 3). Among the over 50 chemokines and 20 chemokine receptors, only a few are thought to Thyroid diseases were diagnosed on the basis of clinical, mark functionally specialized T-cells within the total biochemical and immunological features as well as Th1 and Tc1 T-cell pool (8, 9). from scintiscans. Antibodies against the TSHR (TSH- CXCR6 (Bonzo, STRL33, TYMSTR) (10–12) and its binding-inhibiting immunoglobulin; TBII) and thyroid ligand CXCL16 (SR-PSOX) belong to the few unique peroxidase (TPO) were measured in serum obtained chemokine/receptor pairs that suggest special up to 2 weeks before operation with commercial RIA w immune functions, whereas most other pairs show kits (TRAKhuman DYNOTEST , anti-TPOn DYNO- w promiscuity and overlap in cell and tissue expression. TEST ; Brahms Diagnostica GmbH, Berlin, Germany). CXCR6 seems to mark functionally specialized T-cells The numbers of focal lymphocytic infiltrates and ecto- within the total Th1 and Tc1 cell pool (13). In contrast, pic germinal centers were counted on hematoxilin/ the chemokine receptor is displayed by very few (1–2%) eosin-stained sections of thyroid samples within an 2 Th2 or Tc2 cells. CXCL16 (13–15) is structurally area of 226 mm as described in (21). different from most other chemokines. It is a type I For mRNA quantitation, we used thyroid tissues from transmembrane protein with four distinct domains: GD (n ¼ 16, all females; mean age^S.E.M. 35.6^3.7 one chemokine domain tethered to the cell surface via years), HT (n ¼ 2, both females; 18 and 34 years) a mucin-like stack, which in turn is attached to trans- and TA (n ¼ 11, five females; 55.7^5.2 years) patients membrane and cytoplasmic domains. CXCL16 acts as (same collective as described in detail in (22)). Four an adhesion molecule when expressed on the cell out of these 16 GD patients used for mRNA analysis surface (16). Upon cleavage from the cell surface, the were selected for CXCR6 expression analysis (patients soluble chemokine shows chemoattractive activity (17). 13–16). PBLs from six additional GD patients were Recently published studies indicate a unique role for further included for flow-cytometric analysis (Table 1). CXCR6 and its ligand in attracting activated lympho- All GD patients were treated with anti-thyroid drugs cyte subsets during inflammation, while also facilita- but not corticosteroids at the time of surgery. The dur- ting immune responses via cell contact (16, 18, 19). ation of GD until enrolment was longer than 15 There are no data on the distribution of CXCR6 in months in most cases. Peripheral blood samples from thyroid autoimmunity and few on autoimmune dis- 10 normal adults (two male, eight female; mean age eases at all (20). The aim of our study was to investi- 33.7^2.7 years) without any history of autoimmune gate CXCR6 and CXCL16 mRNA expression in disease were used as controls. The study was approved thyroids affected by GD compared with thyroid tissue by the local Committee of Medical Ethics. from patients with HT and non-autoimmune thyroid autonomy (TA) to gain new insights into the running Isolation of cells and cell lines used immune processes within the thyroid and to character- in this study ize the lymphocytic subpopulations involved. In GD, we isolated thyroid-derived lymphocytes (TLs) and com- Thyroid samples were obtained during operation. TLs pared the expression of CXCR6 with that of peripheral were isolated after mechanical tissue disaggregation

Table 1 Age, sex, duration of disease until enrolment, signs of Graves’ ophthalmopathy (GO), treatment with anti-thyroid drugs and corticosteroids of patients with GD analyzed for CXCR6 expression on PBLs and TLs in ﬂow cytometry.

Patient Patient no. in Duration of Anti-thyroid drug Corticosteroid Signs no. reference (22) Age Sex disease (months) treatment treatment of GO

1 27 Female #15 Yes No Yes 2 22 Female $15 Yes No No 3 49 Male $15 Yes No Yes 4 62 Male $15 Yes No No 5 35 Male $15 Yes No No 6 59 Male $15 Yes No Yes 7 13 18 Female $15 Yes No Yes 8 14 25 Female $15 Yes No Yes 9 15 38 Female #5 Yes No Yes 10 16 50 Female $15 Yes No No

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Downloaded from Bioscientifica.com at 09/27/2021 12:37:12PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2005) 152 CXCR6 in thyroid autoimmunity 637 and Ficoll density-gradient centrifugation. Thyrocytes and (3) CXCR6-PE; CD8-RPE-Cy5; granzyme A-FITC. were enriched after enzymatic digestion of the tissue The cells were analyzed by flow cytometry (FAC- as described in (23). Outgrowing fibroblasts were Scanw; Becton Dickinson, Mountain View, CA, USA) obtained by culturing small pieces of thyroid samples. using electronic gating on lymphocyte subpopulations. PBLs were isolated by Ficoll density-gradient centrifu- The Mann–Whitney test was used to determine statisti- gation. Anaplastic thyroid carcinoma cell lines SW- cal significance. 1736, Hth-74, C-634 (kindly provided by N.-E. Heldin, University of Uppsala, Uppsala, Sweden) and 8505 C, follicular lines FTC-133 and FTC-238 and Real-time reverse transcriptase (RT)-PCR papillary line B-CPAP (DSMZ, Braunschweig, Total cellular RNA was isolated with the Qiagen total Germany), as well as Chinese hamster ovary (CHO) RNA isolation kit (Qiagen GmbH, Hilden, Germany). cells (ATCC) were cultured in Dulbecco’s modified cDNA was synthesized from 1 mgRNAina20ml stan- Eagle’s medium/10% fetal calf serum. dard reaction mixture containing 200 U Superscript II RNaseH reverse transcriptase (Invitrogen). Quantitative Examination of the CXCR6 antibody PCR was performed on a Rotorgene (Corbett Research, Mortlake, Australia) real-time machine by using Since we always measured a lower number of T-cells SYBRw Green I as a double-strand DNA-specific binding expressing CXCR6 than described in (10), the specificity dye and continuous fluorescence monitoring. Amplifi- and sensitivity of the monoclonal antibody (mAb) used cation was carried out in a total volume of 20 ml con- was examined. First, PBLs were cultured with taining 0.5 mM of each primer, 1–3 mM MgCl2,1U 100 ng/ml IL-15 (Immunotools, Friesoythe, Germany) Taq polymerase (Roche), 10 £ Taq buffer (500 mM over 6 days, and the number of CXCR6þ cells was KCl/100 mM Tris/HCl, pH 8.3; Roche), 2 mM of each determined by flow cytometry. Secondly, the CXCR6- dNTP and 1 ml SYBRw Green (1:2000; Molecular encoding cDNA was cloned in the vector pcDNA3.1 Probes Europe, Leiden, The Netherlands) dissolved in Zeo(þ) (Invitrogen GmbH, Karlsruhe, Germany) using DMSO and 1 ml cDNA diluted 1:5. the primer pair 50-CCG GAA GCT TAC CAT GGC AGA The PCR primers were CXCR6 sense (50-CCT GCC GCA TGA TTA CC-30 (sense) and 50-GGA ATT CGA TTA ACC CTG TGC TCT AT-30) and antisense (50-ATG ACA TGC TGG TGG CCT CCA C-30 (antisense). CHO TCA TCC CCC TTG GTT TCA-30), CXCL16 sense (50- cells were transiently transfected with the construct GGG GGC AGT CAC CGC AGT CCT-30) and antisense using Lipofectamine (Invitrogen). mAb binding was (50-ATT AGC CGG GTG TGG TGG TGA GCA-30)or measured after 48 h by flow cytometry. CD18 sense (50-GGC GCA CAA GCT GGC TGA AAA CAA-30) and antisense (50-AGC GCC CGG ATG ACA 0 Analysis by flow cytometry AAC GAC TG- 3 ), respectively. CD18 mRNA was measured in order to quantify leukocytic infiltration Directly fluorochrome-labeled mAbs were supplied by (25). Sizes of PCR products were: CXCR6 459 bp; R & D Systems GmbH (Wiesbaden, Germany; CXCR6- CXCL16 518 bp, and CD18 372 bp. Glyceraldehyde PE, IFN-g-FITC), DakoCytomation Denmark A/S 3-phosphatase dehydrogenase (GAPDH) transcripts (Glostrup, Denmark; CD3-RPE-Cy5, CD8-RPE-Cy5, were measured with a ready-to-use quantitative PCR CD62L-FITC, CD4-FITC), Immunotech (Marseilles, test kit (Roboscreen, Leipzig, Germany) (26). External France; CD45R0-FITC) or BD Biosciences Europe (Hei- cDNA standards were constructed by blunt-end cloning delberg, Germany; CD4-Cy5, CD19-PerCP, granzyme of PCR fragments into pGEM-T vector (Promega, A-FITC). Mannheim, Germany). Plasmid DNA was prepared To enrich intracellular cytokines, cells were treated (Plasmid Mini Kit; Qiagen), quantified and stabilized with PMA (10 ng/ml), ionomycin (1 mmol) and Golgi- in the PCR tubes (27). Amounts were calculated from Plug (1 ml/ml; BD Biosciences) in RPMI 1640/10% a reference curve obtained from the simultaneously fetal calf serum for 4 h. Since ionomycin markedly processed standard curve. Data were expressed as zep- diminishes CXCR6 expression, cells must be pre-stained tomoles (zmol; 10221 mol) of cDNA per attomole with the anti-CXCR6 antibody before treatment (24). (amol; 10218 mol) of GAPDH cDNA. Cells were fixed and permeabilized using Cytofix/ Cytoperm solutions (BD Biosciences) before staining to analyze intracellular cytokines and granzyme A. This Results procedure is not necessary if only cell-surface antigens Flow cytometry are analyzed. Unfixed or fixed PBLs or TLs (3 £ 105) were incubated with mAbs at the desired concentration We first examined whether the available CXCR6 mAb for 30 min at 4 8C. The following mAb combinations was suitable for flow cytometry. IL-15 increased the were used: (1) CXCR6-PE; CD3-RPE-Cy5; CD8-FITC, number of CXCR6þ PBLs by about 5-fold as shown in CD4-FITC, CD45RO-FITC, CD62L-FITC or CD56-FITC; a typical case in Fig. 1A. Moreover, transiently (2) CXCR6-PE; CD8-RPE-Cy5 or CD4-Cy5; IFN-g-FITC transfected CHO cells expressing human CXCR6 were

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CXCR6 expression. All results are summarized in Table 2. Table 2A contains a general overview on the cellular composition of PBLs and TLs. The percentage of memory T-cells (CD3þCD45R0þ) increased in the thyroid compared with periphery. TLs differed from their PBLs counterparts in terms of lymph-node-homing capabilities since CD62L was expressed at only 27% of TLs but 79% of PBLs. The percentage of T-cells capable to produce IFN-g was higher in the thyroid compared the periphery. IFN-gþ cells were more present in CD8þ than CD4þ PBLs and TLs. The expression pattern of CXCR6 within these lymphocytic subpopulations was also characterized (Table 2B, Fig. 2). The percentage of CD3þ and, further subdivided, CD4þ and CD8þ T-lymphocytes expressing CXCR6 in PBLs did not vary between normal subjects and patients with GD. However, these CD4þCXCR6þ and CD8þCXCR6þ T-cells were enriched 3-5-fold in Figure 1 Examination of the CXCR6 mAb. (A) IL-15 increased the the thyroid compared with the periphery. The increase number of CXCR6þ PBLs by about 5-fold. (B) Transiently CXCR6 in CXCR6þ cells was evident within the Th1 þ cDNA-transfected CHO cells were CXCR6 (left, direct-labeled (CD4þIFN-gþ) and Tc1 (CD8þIFN-gþ) T-cell subpopu- CXCR6 mAb; right, indirect detection system). lations (Fig. 2A and B). A higher percentage of peripheral as well as thyroid-derived CD8þ T-cells, com- CXCR6þ. The number of positive cells varied non-sig- pared with CD4þ T-cells, expressed CXCR6. Since the niﬁcantly whether we used a direct or indirect CXCR6 percentage of CXCR6þ T-cells producing IL-4 was labeling system (Fig. 1B). very low in PBLs and TLs (,1%; not shown), we do Secondly, PBLs of normal subjects and GD patients not suggest a function for CXCR6þ Th2 or Tc2 cells as well as TLs of GD patients were analyzed for in the thyroid.

Table 2 CXCR6 expression of TLs and PBLs from patients with GD and normal controls (n ¼ 10) analyzed by ﬂow cytometry using multicolor analysis and lymphocyte gating.

Relative number of cells expressing the marker Marker (%) in each lymphocyte subpopulation expression TLs (GD) PBLs (GD) PBLs (controls)

Characterization of lymphocytes þ þ 100% CD3 CD62L 27.1^6.5 (*P , 0.0001) 79.0^4.6 77.2^2.5 þ CD45RO 61.0^4.5 (*P , 0.02) 42.0^4.8 39.7^2.9 CD4þ 64.4^5.4 65.2^6.5 62.9^3.7 CD8þ 37.1^3.8 33.0^3.3 36.9^3.8 100% CD8þ Granzyme Aþ 47.3^5.3 41.4^5.9 44.0^11.0 þ IFN-g 59.8^6.6 (*P , 0.02) 30.3^4.9 43.9^4.9 þ þ 100% CD4 IFN-g 20.1^3.7 (*P , 0.01) 5.6^0.6 (**P , 0.02) 17.1^2.9 Characterization of CXCR6þ lymphocytes þ þ 100% CD3 CXCR6 21.0^1.9 (*P , 0.0001) 4.7^0.4 4.1^0.5 þ þ þ 100% CD3 CD45RO CXCR6 25.1^2.5 (*P , 0.0001) 8.8^1.7 7.1^0.9 þ þ þ 100% CD3 CXCR6 CD62L 10.5^1.9 (*P , 0.0001) 45.4^6.5 39.8^6.1 100% CD3þCD56þ CXCR6þ 32.1^3.3 32.9^6.7 14.3^3.5 þ þ 100% CD32CD56 CXCR6 8.6^2.2 (*P , 0.05) 3.4^0.7 2.0^0.5 þ þ þ 100% CD3 CD8 CXCR6 28.0^3.4 (*P , 0.0001) 6.5^1.4 6.5^1.1 þ þ þ 100% CD8 IFN-g CXCR6 15.7^2.7 (*P , 0.01) 3.7^1.1 1.5^0.3 þ þ þ 100% CD8 CXCR6 IFN-g 52.6^7.7 (*P , 0.01) 30.4^2.3 44.4^5.7 þ þ þ 100% CD8 granzymeA CXCR6 18.5^4.2 (*P , 0.01) 3.7^0.9 1.5^0.2 100% CD8þCXCR6þ Granzyme Aþ 66.9^7.0 67.9^7.5 80.1^1.4 þ þ þ 100% CD3 CD4 CXCR6 11.7^1.7 (*P , 0.001) 3.4^0.8 2.4^0.2 þ þ þ 100% CD4 IFN-g CXCR6 8.3^2.4 (*P , 0.01) 1.6^0.3 1.2^0.3

*Signiﬁcant differences between TLs and PBLs in GD patients; **signiﬁcant differences between PBLs from GD patients and controls; percentage of labeled cells (means^S.E.M.).

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Figure 2 FACS analysis of PBLs and TLs from the same GD patient. CXCR6 was expressed on a higher percentage of (A) CD4þ and (B) CD8þ TLs than PBLs. (C) The percentage of CXCR6þCD8þ granzyme Aþ cells was also higher in the thyroid compared with the periphery. (D) 60% of peripheral CXCR6þ T-cells failed to express CD62L, whereas approximately 90% of the corresponding thyroid-derived cells lacked CD62L.

Granzymes are important effector molecules for cyto- Most peripheral CD3þCXCR6þ cells have extralym- toxic lymphocytes and induce granule-mediated apopto- phoid tissue-homing potential since 60% failed to sis in target cells. Some 40–50% of CD8þ cells expressed express CD62L. Approximately 90% of thyroid-derived intracellular granzyme A in both PBLs and TLs from GD CXCR6þCD3þ T-cells lack CD62L (Table 2, Fig. 2D). patients. Within these peripheral CD8þ granzyme Aþ Peripheral and thyroid-derived B-cells did not express cells, only a few (,4%) expressed CXCR6, whereas CXCR6 (not shown). nearly 20% carried the receptor in the thyroid (Fig. 2C). The majority of CD8þCXCR6þ peripheral and thyroid-derived cells were positive for granzyme A. CXCR6 and CXCL16 mRNA in thyroid tissues CD3þCD56þ T-cells are effector cells with strong and thyroid-derived cells cytotoxic activity against target cells. In this regard, CXCR6 was expressed on higher percentages of Real-time PCR revealed that thyroid tissues from all GD CD3þCD56þ than on less cytotoxic CD32CD56þ cells. patients with high TPO levels and both HT patients

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Downloaded from Bioscientifica.com at 09/27/2021 12:37:12PM via free access 640 G Aust and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2005) 152 were positive for CXCR6 mRNA, whereas no signal was circulating Th1 and Tc1 cells, is expressed on a present within some tissues from GD patients with low higher percentage of TLs than PBLs from GD patients. or no TPO levels and TA tissues (Fig. 3A). We found a The data raise the question of the CXCR6’s function slight correlation between CXCR6 and (1) CD18 mRNA in the thyroid. The result agrees with our own previous levels (r ¼ 0.51; P ¼ 0.044) and (2) TPO antibody study on CXCR3 and CCR5 (29), chemokine receptors levels (r ¼ 0.66; P ¼ 0.05) in GD patients. There was that are thought to be predominantly, but not exclu- no correlation between CXCR6 mRNA levels and TBII sively, found on Th1 cells. Both receptors were (P ¼ 0.42) and the number of lymphocytic infiltrates expressed on a higher percentage of TLs compared (P ¼ 0.60) or germinal centers (P ¼ 0.60) in these with PBLs, and may be involved in the recruitment of patients. We found no difference in CXCR6 mRNA lymphocytes to the thyroid in GD (30, 31). We suggest levels in GD compared with normal TA tissue. Lowest that, in addition to CXCR3 and CCR5, CXCR6 may be a levels were present in TA nodules. Here, three out of marker for cells that have migrated into thyroid tissue nine samples were negative for CXCR6 mRNA. and assist there, independently of the bias of the under- Using simple RT-PCR, PBLs and TLs were CXCR6 lying disease. This is confirmed by the fact that most of mRNA-positive, whereas no signal was obtained from the CXCR6þ T-cells in the thyroid lost their lymph- thyroid-derived fibroblasts (Fig. 3C). Thyrocytes node-homing capabilities since they were CD62L2. showed only a very faint signal. Thyroid carcinoma The antigen is downregulated in response to migration cell lines were positive for CXCR6 mRNA. The and activation by proteolytically cleaving from the cell expression level varied considerably. surface (32). Moreover, our assumption is supported Quantifying CXCL16 cDNA in thyroid tissues by recent studies demonstrating that CXCR6þ T-cells (Fig. 3B), the levels were somewhat high compared were enriched in tissue sites of inflammation, such as with other chemokines quantified (21, 25, 28). Surpris- rheumatoid joints and inflamed livers (13, 18). ingly, CXCL16 cDNA levels within the two HT tissues Although a higher percentage of TLs compared with were within the same range as those of many GD and PBLs were CXCR6þ in GD, we did not find a difference normal TA tissues. This suggests that cells other than between CXCR6 mRNA levels in the thyroid tissues the known CXCL16 producers such as dendritic cells from different patients groups. Mistakes in CXCR6 and macrophages may express CXCL16 mRNA. mRNA determination were unlikely since quantitation Indeed, besides TL, thyrocytes were strongly positive of the chemokine receptor/ligand pair CXCR5/CXCL13 for this chemokine mRNA (Fig. 3C). Thus, the in the same cDNAs samples resulted in significantly CXCL16 mRNA levels tend to represent the presence higher levels in GD compared with TA samples and of this chemokine within several cell types of the thyroid the highest levels in HT tissues (21). Our results than a specific expression by immunocompetent cells. suggest that the number of CXCR6þ and thus Th1 and Tc1 cells in HT may not be higher than in other thyroids, including non-autoimmune TA. There are Discussion several possible explanations for this result. As in GD tissues, the chronic lymphocytic infiltrate does not We have demonstrated here that CXCR6, a chemokine reflect the initiation, but rather the maintenance or receptor present on a small subset of specialized perturbation, of the immune process in HT tissues

Figure 3 Quantitation of (A) CXCR6 and (B) CXCL16 mRNA in thyroid tissues of GD patients with high (.1000 U/ml; TPOhigh; n ¼ 8) and low or no antibodies against TPO (TPOlow; n ¼ 8), HT patients (HT; n ¼ 2) and in TA patients (normal and nodular tissue; n ¼ 11), *P , 0.005. (C) Simple RT-PCR for CXCR6 and CXCL16 mRNA in thyroid- derived cell populations (thyrocytes, ﬁbroblasts, lymphocytes ¼ TLs), thyroid carcinoma cell lines and PBLs.

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Downloaded from Bioscientifica.com at 09/27/2021 12:37:12PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2005) 152 CXCR6 in thyroid autoimmunity 641 since a substantial number of thyrocytes are always chemokine/receptor pair in the thyroid? CXCL16 not destroyed at the time of surgery (33, 34). The time of only attracts CXCR6 expressing T-cells, natural killer action of specialized Th1 and Tc1 cells may be over (NK) cells and NK/T-cells to the site of inflammation during HT. This assumption is confirmed by reports and injury, but also mediates firm adhesion of showing a significant increase in IFN-g-inducible che- CXCR6-expressing cells in its transmembrane form mokines CXCL10 (IP-10) and CXCL9 (Mig) strongly (16, 19). The chemokine is induced by pro-inflamma- associated with Th1-mediated immune responses in tory stimuli such as tumour necrosis factor-a and thyroid tissue specimens obtained from subjects IFN-g (17). Surprisingly, we did not find any differences affected by recent-onset GD (2, 35, 36). An increase between CXCL16 mRNA levels in the thyroid tissues in serum CXCL10 levels has only been found in analyzed. Normally, CXCL16 is only expressed by newly diagnosed GD patients, compared with healthy macrophages, dendritic cells and CD19þ B-cells (14, subjects and patients with long-standing disease. 42). In our study, a strong CXCL16 mRNA signal was An inverse correlation between circulating CXCL10 obtained from thyrocytes and thyroid carcinoma cell levels and disease duration has been demonstrated lines. Thus, CXCL16 also seems to be produced by epi- (37). This means that changes in the composition thelial cells, which confirms older studies showing that of regulating T-cell subsets involved, and thus thyrocytes were potential cytokine producers (43). chemokines and their receptors, occur within the Untreated thyrocytes secrete CXCL8 (IL-8) and the thyroid but also at the periphery, depending on the other chemokine that binds to the CXCR2, CXCL1 duration of GD. (GRO-a) (28). CCL2 (monocyte chemoattractant pro- Data on CXCR6 in autoimmunity are very limited tein-1; MCP-1) is a chemokine that attracts T-lympho- (20). An increase of CXCR6þ cells was shown in cytes as well as monocytes, and can be also produced arthritic synovial fluid, which was discussed as playing by thyrocytes (44). Thyrocytes from autoimmune thyr- a role of the receptor in the traffic of effector T-cells that oid disorders produce the chemokines CXCL9 and mediate type 1 inflammation. The paucity of data CXCL10 after stimulation (36). All these studies suggest published on CXCR6 may be due to the discrepant a possible role for thyrocytes on accumulation of leuko- results obtained with the available antibodies and the cytes in the tissue from the blood. Unfortunately, useful first data published (10). The percentages of CXCR6þ CXCL16 antibodies for immunohistology or establish- T-cells in normal subjects in our study were lower, ment of protein screening assays were not available. although we used the same mAb and staining tech- In summary, our study demonstrates that the per- nique as in (10) (15 versus 4.1% CXCR6þ cells centages of Tc1 (CD8þIFN-gþ) and Th1 (CD4þIFN- within CD3þ). Thus, we first showed that the mAb gþ) cells as well as cytotoxic effector cells (CD8þ used indeed detects CXCR6 and that the percentage of granzymeAþ) expressing CXCR6 were increased in CXCR6þ T-cells does not depend on direct or indirect TLs compared with PBLs in GD patients. However, we labeling methods. Moreover, IL-15 increased the did not find any difference between CXCR6 mRNA number of CXCR6þ PBLs by about 5-fold, which was levels in thyroid tissues from different patient groups. in accordance to data obtained by Unutmaz et al. (10). CXCR6 could be a marker for cells that have migrated CXCR6þ T-cells are thought to represent type 1 cells, into the thyroid and assist there, independently of the which mainly regulate cellular but not humoral bias of the underlying disease. immune responses. This might explain the missing correlation of CXCR6 mRNA with TBII and the slight correlation of CXCR6 mRNA with TPO antibodies. TPO Acknowledgements antibodies better reflect the immune responses in long-standing disease compared with TBII (38). The authors are thankful to D Sittig for her excellent Additionally, thyroid autoantibodies might not be pro- technical assistance. This study was supported by the duced exclusively by the thyroid itself (39–41). Deutsche Forschungsgemeinschaft (project AU-132-2). The correlation between CXCR6 mRNA and CD18 mRNA is weak. CD18 mRNA also includes the mRNA of those lymphocytes that were not located within infil- References trates or germinal centers, but scattered throughout the tissue and, additionally, the mRNA of non-lymphoid 1 Mosmann TR, Li L & Sad S. Functions of CD8 T-cell subsets secret- leukocytes, the macrophages. We do not know where ing different cytokine patterns. Seminars in Immunology 1997 9 þ 87–92. CXCR6 lymphocytes are located within Graves’ 2 Rotondi M, Lazzeri E, Romagnani P & Serio M. Role for interferon- thyroids or whether or not they accumulate on gamma inducible chemokines in endocrine autoimmunity: an certain structures. This may explain why we missed a expanding field. Journal of Endocrinological Investigation 2003 26 correlation between CXCR6 and defined lymphocytic 177–180. 3 Dogan RN, Vasu C, Holterman MJ & Prabhakar BS. Absence of infiltrates and germinal centers. 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