A Novel Model for Lymphocytic Infiltration of the Thyroid Gland Generated by Transgenic Expression of the CC CCL21 This information is current as of September 25, 2021. Andrea P. Martin, Elizabeth C. Coronel, Gen-ichiro Sano, Shu-Cheng Chen, Galya Vassileva, Claudia Canasto-Chibuque, Jonathon D. Sedgwick, Paul S. Frenette, Martin Lipp, Glaucia C. Furtado and Sergio A. Lira

J Immunol 2004; 173:4791-4798; ; Downloaded from doi: 10.4049/jimmunol.173.8.4791 http://www.jimmunol.org/content/173/8/4791 http://www.jimmunol.org/ References This article cites 61 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/173/8/4791.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Model for Lymphocytic Infiltration of the Thyroid Gland Generated by Transgenic Expression of the CC Chemokine CCL211

Andrea P. Martin,2* Elizabeth C. Coronel,2† Gen-ichiro Sano,* Shu-Cheng Chen,† Galya Vassileva,† Claudia Canasto-Chibuque,* Jonathon D. Sedgwick,‡ Paul S. Frenette,* Martin Lipp,¤ Glaucia C. Furtado,2* and Sergio A. Lira3*

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin Downloaded from promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or -␣ but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles http://www.jimmunol.org/ in the thyroid. The Journal of Immunology, 2004, 173: 4791Ð4798.

he autoimmune thyroid diseases (AITD)4 are the most autoantibodies directed against thyroid Ags such as thyroglobulin common autoimmune endocrine diseases, affecting (TG), thyroid peroxidase, and the thyroid-stimulating hormone T ϳ1.5% of the general population (1). The major forms of (TSH) receptor (TSH-R) (2). These autoantibodies can either stim- AITD are Hashimoto’s thyroiditis (HT), causing hypothyroidism, ulate or inhibit thyroid function. and Graves’ disease (GD), causing hyperthyroidism. The hallmark Despite substantial clinical and experimental data supporting a of these conditions is thyroid dysfunction associated with the pres- role for lymphocytes in triggering and sustaining AITD, factors ence of lymphocytic infiltrates in the thyroid (2, 3). In HT, there is responsible for their recruitment and retention in the thyroid remain by guest on September 25, 2021 a diffuse lymphocytic infiltration with the presence of macro- uncertain. Migration of T cells appears to be regulated by inflamma- phages and destroyed thyrocytes. The lymphocytes often cluster to tory (inducible) and constitutive . Examples of inflamma- form lymphoid follicles with germinal centers. Lymphocytic infil- tory chemokines include CXCL8, CCL2, CCL3, CCL4, and trates, usually without germinal center formation, are also seen in CXCL10. Expression of these chemokines occurs during inflam- GD (4), but diffuse follicular cell hyperplasia and increased vas- mation and is required for attraction of specific leukocyte subsets cularity, rather than thyrocyte destruction, are observed (3). mediating inflammatory reactions (6, 7). CXCL10, the best studied The etiopathogenic role of lymphocytes in HT and GD has been of the inflammatory chemokines regulating T cell migration, is in- amply studied and a variety of effector mechanisms have been ␥ defined for both T and B cells (2, 5). T cells cause thyroid de- duced by IFN- , a key involved in inflammation and im- struction directly, via cytotoxicity, or indirectly, through mune regulation (8). CXCL10 interacts with CXCR3, a chemokine that induce apoptosis of thyroid follicle cells (5). B cells produce receptor expressed by effector T cells and endothelial cells (9). Chemokines constitutively produced by lymphoid organs (CCL19, CCL21, CXCL12, and CXCL13) are involved in physi- *Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029; ological trafficking of leukocytes and their segregation into spe- †Schering-Plough Research Institute, Kenilworth, NJ 07033; ‡DNAX Research, Palo cialized compartments. Among these, CCL19 and CCL21 are the Alto, CA 94304; and §Max-Delbruck-Center for Molecular Medicine, Berlin, Germany best-characterized chemokines regulating homeostatic T cell mi- Received for publication June 4, 2004. Accepted for publication July 30, 2004. gration. Both chemokines interact with a common receptor, CCR7, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance which is expressed by naive and memory T cells (10). CCL21 and with 18 U.S.C. Section 1734 solely to indicate this fact. CCL19 are displayed by high endothelial venules (HEVs) and ac- 1 S.A.L. is an Irene Diamond Associate Professor of Immunology. This work was tivate integrins after interaction with CCR7 present on circulating supported in part by a grant from the Irene Diamond Fund, and by Grants DK 067989 lymphocytes (11). The functional consequence of integrin activa- (to S.A.L.) and HL 69438 (to P.S.F.) from the National Institutes of Health. tion is increased adhesiveness of lymphocytes to the endothelium, 2 A.P.M., E.C.C., and G.C.F. contributed equally to the manuscript. a key step in lymphocyte transendothelial migration. Accordingly, 3 Address correspondence and reprint requests to Dr. Sergio A. Lira, Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1630, New mice lacking CCR7, CCL19, and CCL21 have defective homing of York, NY, 10029-6574. E-mail address: [email protected] T cells into lymphoid tissue (12–14). In addition to controlling the 4 Abbreviations used in this paper: AITD, autoimmune thyroid diseases; HT, Hashi- initial aspects of lymphocyte migration, chemokines also control moto’s thyroiditis; GD, Graves’ disease; TG, thyroglobulin; TSH, thyroid-stimulating hormone; HEV, high endothelial venule; Lt␣, lymphotoxin ␣; PNAd, peripheral targeted homing of T and B cells within lymphoid organs. The lymph node addressin; LDL, low density lipoprotein. specific segregation of B and T cells in secondary lymphoid organs

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 4792 TRANSGENIC EXPRESSION OF CCL21 IN THE THYROID is dependent on multiple chemokine gradients established by den- River Laboratories, Wilmington, MA) according to published procedures dritic cells, stromal cells, and lymphocytes (15). (33). Genotyping of the TGCCL21 transgenic founders and progeny was Chemokines, with the exception of CXCL12 (16), are not ex- conducted by PCR analysis of mouse tail DNA using recognition primers for the rat TG promoter 5Ј-CTG CAG ACA AGC AGG CAT GCA-3Ј pressed by the normal human thyroid at appreciable levels. How- (forward) and 5Ј-CAC ACA TGG CAC ATA TGC-3Ј (reverse) as previ- ever, most chemokines studied to date are expressed within the ously described (34). The endogenous low density lipoprotein (LDL) thyroids of GD and HT patients. These include CXC chemokines, was used as an internal control using primers LDLL 5Ј-CGC AGT GCT CXCL1 (17), the IFN-␥ inducible chemokines CXCL9, and CCT CAT CTG ACT TGT-3Ј (forward) and LDLU 5Ј-ACC CCA AGA CGT GCT CCC AGG ATG A-3Ј (reverse). PCR conditions were 94°C, CXCL10 (9, 18, 19), CXCL12 (16), and CXCL13 (20). The CC 30 s; 60°C, 30 s; 72°C, 60 s for 30 cycles. chemokines CCL2, CCL3, CCL4, and CCL5 are also reported to TGCCL21 transgenic mice (line 24) were crossed to CCR7Ϫ/Ϫ mice, be expressed in thyroid disease (19, 21, 22). Thyroid expression of described by Forster et al. (12), to LT␣Ϫ/Ϫ (35) and to RAG-2Ϫ/Ϫ mice these chemokines may be critical for the recruitment of chemokine (36). Mice hemizygous for CCL21 and null for RAG or CCR7 or LT␣ (referred to as TGCCL21/RAGϪ/Ϫ mice, TGCCL21/CCR7Ϫ/Ϫ mice, and receptor-expressing leukocytes to the thyroid. Indeed, CXCR3, Ϫ Ϫ TGCCL21/LT␣ / mice, respectively) and littermate controls were used CCR2, and CCR5 expression is elevated on infiltrating inflamma- for the experiments described here. Transgenic mice expressing GFP (37) tory cells relative to peripheral blood leukocytes (9, 18, 21, 23). and L-selectinϪ/Ϫ mice (38) were backcrossed over 10 generations in the The cellular source of these chemokines in the thyroid is usually C57BL/6 background. All mice were housed under specific-pathogen-free the infiltrating lymphocytes, but thyrocytes and fibroblasts also conditions in individually ventilated cages at the Mount Sinai School of Medicine Animal Facility. All experiments were performed following in- express chemokines in diseased thyroid (17). Different proinflam- stitutional guidelines. matory stimuli such as IL-1, IFN-␥, and TNF-␣ increase expres- sion of chemokines by thyroid follicular cells in culture (17, 18, Downloaded from Histology 24, 25), suggesting that inflammatory cytokines may trigger ex- pression of chemokines by thyrocytes and other resident thyroid Tissues for light microscopic examination were fixed by immersion in 10% cells in vivo. Interestingly, recent studies have shown that chemo- phosphate-buffered formalin and then processed for paraffin sections. Rou- tinely, 5-␮m sections were cut and stained with H&E. For immunohisto- kines required for the formation and maintenance of lymphoid fol- chemical staining, fresh frozen sections were first fixed with ice-cold ac- licles (i.e., CXCL12, CCL21, and CXCL13) are expressed in the etone for 20 min, dried, and stored at Ϫ20°C. Slides were stained and thyroids of AITD patients (20, 26). The expression of these mol- analyzed as described previously (39). Slides were incubated for1hat http://www.jimmunol.org/ ecules in the thyroid suggests that chemokines may be necessary room temperature with purified primary Abs followed by incubation with for recruitment and subsequent organization of lymphoid follicles the appropriate labeled secondary Abs for 30 min. Primary Abs used were anti-Thy1.2 (53-2.1), CD3 (145-2C11), CD4 (H129.19), B220 (RA3-6B2), in the thyroid. CCL21 and CXCL13 have been shown to induce peripheral lymph node addressin (PNAd) (MECA79) from BD Biosciences formation of lymphoid structures when expressed in the pancreas (San Diego, CA), and anti-6Ckine/CCL21 (no. AF457) from R&D Systems (27–29). However, this property does not seem to be general, as (Minneapolis, MN). Secondary Abs used were Alexa Fluor 594 donkey expression of CCL21 in brain and skin of transgenic mice fails to anti-goat (no. A-11058), Alexa Fluor 488 and 594 goat anti-rat IgM (nos. A-21212 and A-21213) and anti-rat IgG (nos. A-11006 and A-11007) from induce development of such structures (30). Here, we directly test Molecular Probes (Eugene, OR) and FITC and rhodamine red-X goat anti- the hypothesis that the chemokine CCL21 may be directly impli- Armenian hamster (nos. 127-095-160 and 127-295-160) and Cy5 goat anti- cated in regulating influx of lymphocytes into the thyroid. We rat (no. 112-175-167) from Jackson ImmunoResearch Laboratories (West by guest on September 25, 2021 show that CCL21 is sufficient to drive not only influx, but also Grove, PA). formation of lymph node-like structures in the thyroid of trans- genic animals, suggesting that this chemokine and its receptor, Flow cytometry CCR7, may be involved in the formation of lymphoid follicles in To prepare single cell suspension, individual thyroids from TGCCL21 an- the thyroid of patients with AITD. Additionally, we show that imals (6 wk of age) were minced in serum-free RPMI 1640 media con- infiltration of the thyroid by lymphocytes under these conditions is taining 0.0012 U/ml dispase I (Sigma-Aldrich, St. Louis, MO) and 0.25 independent of L-selectin and lymphotoxin ␣ (LT␣). U/ml collagenase type II (Worthington Biochemical/Invitrogen Life Tech- nologies, Rockville, MD), incubated for2hat4°C followed by an addi- tional incubation of 10 min at 37°C. Thyroid cell suspensions obtained Materials and Methods from both lobes were centrifuged at 250 ϫ g for 10 min at 4°C and re- Construction of the TGCCL21 transgene suspended in FACS staining buffer (PBS containing 2% FCS and 0.01% sodium azide). The rat TG promoter (GenBank X06162) was amplified by PCR from rat Cells were incubated for 20 min at 4°C with 5 ␮g/ml Fc block (BD genomic DNA using recognition primers that contained sites for HindIII at Pharmingen, San Diego, CA) and then stained with directly conjugated Ј Ј Ϫ Ϫ the 5 end and for EcoRI at the 3 end to include nucleotides 868 to 1 primary mAbs. mAbs to the following mouse leukocyte surface markers from the transcription start site. The rat TG promoter was subcloned into were purchased from BD Pharmingen: CD45 (30-F11), CD3e (145-2C11), HindIII/EcoRI sites of pBS plasmid (Stratagene, La Jolla, CA) to generate CD4 (RM4-5), B220 (RA3-6B2), CD62L (MEL-14), CD11c (HL-3), and the TG/pBS plasmid. CD11b (M1/70). To determine viability, samples were subsequently A BstX I fragment (2.4 kb) containing CCL21 genomic DNA encoding stained with 20 ␮lof5␮g/ml propidium iodide (Calbiochem, La Jolla, CCL21a (6Ckine serine) was isolated from a BAC clone (31) and blunt-end CA). Events were acquired on a BD Biosciences FACScan and analyzed ligated into the EcoRI site of the TG/pBS plasmid to generate a vector using CellQuest software. To rule out the presence of rare thymic contam- encoding the TGCCL21 transgene. The TGCCL21 transgene (3.3 kb) was inants in the thyroid single cell preparations, we routinely determined the released from the TG/pBS vector by digestion with XhoI/XbaI restriction relative proportion of thymocytes or immature T cells (CD4ϩ/CD8ϩ cells). enzymes. Separation of the transgene DNA from vector DNA was accom- Samples that contained Ͼ2% of this cell population were excluded from plished by zonal sucrose gradient centrifugation as described (32). The the analysis. fractions containing the transgene DNA were pooled, microcentrifuged through Microcon-100 filters (Millipore, Bedford, MA), and washed five times with microinjection buffer (5 mM Tris-HCl, pH 7.4, 5 mM NaCl, 0.1 Adoptive cell transfers mM EDTA). Spleen cells (107) were resuspended in PBS and injected i.v. Lymph nodes Mice and thyroids from recipient animals were collected at various points (24, 48, 72, and 96 h) and fixed in 1.5% paraformaldehyde containing 20% The TGCCL21 transgene DNA was resuspended in microinjection buffer sucrose for 24 h at 4°C. Fixed tissues were frozen in OCT compound to a final concentration of 1–5 ng/␮l, microinjected into fertilized eggs (Tissue Tek, Torrance, CA), cryosectioned (8 mm), and examined under a ϫ from (C57BL/6J DBA/2)F2 females (The Jackson Laboratory, Bar Har- fluorescent microscope. In some instances, images were overlaid and pro- bor, ME), and transferred into oviducts of ICR foster mothers (Charles cessed using Adobe Photoshop. The Journal of Immunology 4793

Results TGCCL21 mice express CCL21 in the thyroid CCL21 is expressed in the thyroid of patients with AITD (20, 26), but it is unclear whether it has any role in pathogenesis. To inves- tigate whether CCL21 could direct the migration of leukocytes to the thyroid, we targeted its expression to the thyroid of transgenic mice. To promote expression of CCL21 in the thyroid, we con- structed a transgene (TGCCL21) in which the CCL21 gene was placed downstream of a segment of the rat thyroglobulin promoter (Ϫ868 to Ϫ1 bp from the transcription start site). This promoter directs the expression of transgenes specifically to thyroid follic- ular cells (40–44). Five transgenic lines were derived from a total of 17 transgenic founders identified by PCR analysis of genomic DNA. TGCCL21 transgenic mice developed normally and were fertile. Transgenic lines were screened for expression of CCL21 in the thyroid by Western blot (not shown). Two lines (nos. 24 and 26) were selected and expanded for the analyses presented here. The expression of CCL21 in the thyroid was analyzed by im- Downloaded from munohistochemical staining using anti-CCL21 Abs (Fig. 1, A and B). We detected CCL21 immunoreactivity in thyroid follicular cells, within the colloid material and in the thyroid parenchyma (Fig. 1B), suggesting that follicular cells secrete CCL21, as ob- served in other transgenic experiments (30). No immunoreactivity for CCL21 was observed in the control thyroids (Fig. 1A). http://www.jimmunol.org/

CCL21 promotes recruitment of mononuclear cells into the thyroid To determine whether CCL21 induces lymphocyte recruitment to the thyroid, we examined H&E-stained paraffin sections of the thyroid of transgenic TGCCL21 mice and their control littermates. No infiltrates were observed in thyroid sections from wild-type littermates (n ϭ 5) (Fig. 1C). However, mononuclear infiltrates were observed in the thyroid of 5 of 8 founders examined (ages 74–208 days old). The by guest on September 25, 2021 infiltrates were composed of mononuclear cells and varied in size from mild (Ͻ20% involvement) to severe (Ͼ50% involvement). Analysis of thyroid sections from transgenic mice in line 24 (16–250 FIGURE 1. Expression of CCL21 in the thyroid induces development days old) also showed mononuclear infiltrates (Fig. 1D) in most (13 of lymphoid aggregates and HEVs. A, CCL21 immunostaining of wild- of 17) of the animals analyzed. Immunohistochemical staining using type thyroid and (B) TGCCL21 thyroid. Notice the presence of CCL21 Abs against cell surface markers showed that both T and B lympho- immunoreactive material in the thyroid follicles of TGCCL21 mice. C, cytes were present in the cellular infiltrates (Fig. 1E) and appeared to H&E-stained paraffin section of wild-type thyroid and (D) TGCCL21 thy- roid showing cellular infiltrates in the parenchyma. E, Cell infiltrates in the be organized into separate clusters throughout the thyroid infiltrates thyroid were analyzed by immunohistostaining with anti-Thy1.2 (green) and (Fig. 1F). Occasionally, germinal center-like structures were observed anti-B220 (blue) Abs. Notice the topological segregation of T cells (green) and Ͼ in the thyroid of aged mice ( 300 days), but no significant mitotic B cells (blue) (F). G, H&E-stained paraffin section of TGCCL21 thyroid show- activity was observed within these structures (not shown). Scattered ing a prominent vascular structure with cuboidal endothelium. Notice the pres- throughout the transgenic thyroid, we observed HEVs (Fig. 1G) ence of RBC and mononuclear cells within the lumen of the vessel (arrow). In expressing PNAd (Fig. 1H), an Ag marking the presence of L-selectin H, expression of the adhesion molecule PNAd, detected by immunohisto- ligands in lymph nodes. Taken together, these results indicate that chemistry, in vessels (arrows) similar to the one shown in G. Original mag- expression of CCL21 within the thyroid promotes accumulation of T nification: AÐE, ϫ100; F, ϫ200; GÐH, ϫ400. and B cells and development of HEVs. CD4ϩ T cells (Fig. 2). B cells (B220ϩ) accounted for ϳ27% of the Thyroid infiltrates are composed of naive T and B cells infiltrates and the remaining 3% of the cells were dendritic cells. Histological analysis of the thyroid of TGCCL21 animals (6 wk of Most of the T cells expressed high levels of L-selectin (CD62L), age) revealed a moderate mononuclear cell infiltration (20–50% of suggesting a naive phenotype. the thyroid occupied by mononuclear cells). To further character- ize the composition of these infiltrates, we performed flow cytom- CCL21 induces thyroid lymphocyte recruitment through CCR7 etry analyses using Abs against several leukocyte cell surface Expression of CCL21 is a major factor regulating recruitment of markers. Single cell suspensions of thyroids from control and naive and memory T cells into lymphoid tissues (46). Although transgenic mice were prepared according to the method described there are at least three known receptors in mice for CCL21 (CCR7, by Caturegli et al. (45). A small fraction of CD45ϩ cells were CXCR3, and an unclassified receptor referred to as CCX-CKR (47, found in the control thyroids (Ͻ2% of total cells, n ϭ 4). In con- 48)), recent data suggest that the main receptor mediating CCL21- trast, CD45ϩ cells represented 30–50% of all cells in the thyroid induced lymphocyte recruitment into lymphoid organs is CCR7 of TGCCL21 animals at 6 wk of age (n ϭ 10). The CD45ϩ cells (46). To examine whether CCR7 mediates the recruitment of lym- were mostly (ϳ70%) T cells (CD3ϩ), the majority of which were phocytes into the thyroid, we bred CCR7Ϫ/Ϫ mice with TGCCL21 4794 TRANSGENIC EXPRESSION OF CCL21 IN THE THYROID

FIGURE 2. Naive lymphocytes infiltrate the thyroid of TGCCL21 ani- mals. Single cell suspensions of the thyroid of TGCCL21 mice (6 wk old) were stained with the indicated Abs. Cells were gated on the viable leu- kocyte population (CD45ϩ/PIϪ). Cells in the histogram plot were gated on the CD45ϩ/PIϪ/CD3ϩ subset. Results are representative of several inde- Downloaded from pendent experiments (n ϭ 10 mice); PI, propidium iodide. FIGURE 4. Absence of cellular infiltrates and HEV structures in TGCCL21/RAGϪ/Ϫ mice. A, Large cellular infiltrates in TGCCL21/ RAGϩ/ϩ thyroid. B,Noinfiltrates are observed in TGCCL21/RAGϪ/Ϫ mice to generate animals that expressed CCL21 in the thyroid, but thyroid. PNAd staining is observed in HEVs in the thyroid of TGCCL21/ ϩ/ϩ Ϫ/Ϫ lacked CCR7 expression (TGCCL21/CCR7Ϫ/Ϫ mice). Histologi- RAG (C), but absent in the thyroid of TGCCL21/RAG mice (D). ϫ ϫ cal examination of thyroids from TGCCL21/CCR7ϩ/ϩ or Original magnification: AÐB, 200; CÐD, 400. ϩ Ϫ TGCCL21/CCR7 / mice (n ϭ 10 mice/group) revealed infiltra- http://www.jimmunol.org/ tion by mononuclear cells (Fig. 3A). In contrast, none of the thy- roids from TGCCL21/CCR7Ϫ/Ϫ mice (n ϭ 10) examined had leu- HEV formation and that factors produced by the infiltrating lym- kocytic infiltrates when examined by light (Fig. 3B)or phocytes and/or dendritic cells may be important for the develop- immunofluorescence microscopy using Abs against the pan-leu- ment of these structures in the tissue. kocyte marker CD45 (not shown). These findings indicate that Expression of CCL21 promotes recruitment of adoptively CCR7 is the main mediating CCL21-induced transferred lymphocytes to the adult thyroid lymphocyte recruitment into the thyroid. To test whether CCL21 expression could promote recruitment of ϩ CCL21 is not sufficient to promote HEV development in the donor lymphocytes to the thyroid, we transferred 107 GFP by guest on September 25, 2021 thyroid splenocytes (i.v.) into the TGCCL21/RAGϪ/Ϫ animals described ϭ ϩ HEVs, specialized blood vessels used by circulating lymphocytes above (n 8). GFP splenocytes were also injected into control RAGϪ/Ϫ mice (n ϭ 5) that did not express CCL21 in the thyroid to migrate into lymphoid organs, were present in the thyroid pa- ϩ renchyma of TGCCL21 mice. Next, we asked whether CCL21 (Fig. 5B). Five days later we examined the presence of GFP cells in frozen sections of lymph nodes, spleen, and thyroids of could directly promote the development of HEVs in this tissue. To Ϫ/Ϫ Ϫ/Ϫ ϩ this end, we crossed the TGCCL21 mice with RAG-2Ϫ/Ϫ mice, TGCCL21/RAG and RAG mice. As expected, GFP cells which are devoid of mature T and B cells and examined the pres- accumulated in lymph nodes (Fig. 5A, a and b) and spleens (not shown) of both TGCCL21/RAGϪ/Ϫ and RAGϪ/Ϫ mice. Interest- ence of HEVs in the thyroid (Fig. 4). As expected, significant ϩ ϩ/ϩ ingly, clusters of GFP cells were visualized in the thyroid of the infiltrates were observed in the thyroid of TGCCL21/RAG Ϫ/Ϫ ϭ TGCCL21/RAG mice (Fig. 5C), but not in the thyroid of mice (n 5) whereas no infiltrates were observed in the thyroid of Ϫ/Ϫ TGCCL21/RAGϪ/Ϫ mice (n ϭ 8), by H&E staining (Fig. 4, A and RAG mice (Fig. 5B) 5 days after transfer. Most of the infil- trating GFPϩ cells at this time point were CD4ϩ T cells (not B, respectively) or CD45 staining (not shown). HEVs expressing ϩ PNAd (Fig. 4C), were found within TGCCL21/RAGϩ/ϩ thyroids. shown). Sixty days after transfer, large clusters of GFP cells were ϭ In contrast, no HEVs were observed in the TGCCL21/RAGϪ/Ϫ observed in the thyroid (Fig. 5D)(n 2). These cells were mostly thyroid (Fig. 4D), suggesting that CCL21 is not sufficient to drive T and B cells that appeared to segregate into specific areas (Fig. 5E). Within these clusters we observed PNAdϩ vessels (Fig. 5F). These results indicate that expression of CCL21 by thyroid follic- ular cells promotes mobilization of adoptively transferred lympho- cytes into the thyroid parenchyma.

Expression of CCL21 promotes the recruitment of adoptively transferred lymphocytes independently of HEV formation and L-selectin expression The absence of both lymphoid infiltrates and HEVs in TGCCL21/ RAGϪ/Ϫ suggested that the presence of HEVs was important for lymphocytic recruitment. To test this possibility, we transferred Ϫ/Ϫ FIGURE 3. Absence of cellular infiltrates in TGCCL21/CCR7Ϫ/Ϫ mice. donor wild-type splenocytes into TGCCL21/RAG mice and A, Large cellular infiltrates in TGCCL21/CCR7ϩ/Ϫ thyroid. B,Noinfil- examined thyroid specimens for the presence of HEVs and expres- trates are observed in TGCCL21/CCR7Ϫ/Ϫ thyroid. Original magnifica- sion of PNAd. We found no HEV-like structures, or PNAd ex- tion: AÐB, ϫ200. pression up to 96 h after adoptive transfer, indicating that HEVs The Journal of Immunology 4795 Downloaded from http://www.jimmunol.org/ FIGURE 5. Selective migration of adoptively transferred GFPϩ splenocytes into the thyroid of mice expressing CCL21. GFPϩ splenocytes were transferred i.v. into RAGϪ/Ϫ mice and into TGCCL21/RAGϪ/Ϫ mice. GFPϩ splenocytes migrated to the lymph nodes of RAGϪ/Ϫ (Aa) and TGCCL21/ RAGϪ/Ϫ(Ab) mice with similar efficiency. RAGϪ/Ϫ mice do not express CCL21 in the thyroid (B), but the TGCL21/RAGϪ/Ϫ mice do express it (red, C). Five days after the adoptive transfer, GFPϩ cells (green) colocalized with CCL21 expressing cells (red) in the thyroid of the TGCL21/RAGϪ/Ϫ mice (C), but not in the thyroid of RAGϪ/Ϫ mice (B). A large cluster of GFPϩ cells 60 days after transfer (D), with T cells present predominantly in the right part of the infiltrate (yellow) and B cells (blue) in the left side (E). Expression of PNAd (red) is associated with the presence of large clusters of GFPϩ cells (F). Shown in the inset of F is a higher magnification of the PNAd-positive vessel marked by the arrow. Original magnification: A, ϫ40; BÐF, ϫ100; inset in F, ϫ400. by guest on September 25, 2021 are not required for initial colonization of the thyroid by lympho- 2.8 ϫ 103 cells vs 2.2 ϫ 103 cells, n ϭ 3). These results indicate cytes (data not shown). that L-selectin is not required for initial entry of lymphocytes into The absence of PNAd expression suggested that the initial lym- the thyroid of TGCCL21/RAGϪ/Ϫ mice. phocyte infiltration might have occurred through an L-selectin- Lymphocyte infiltration and topological arrangement in independent pathway. To test this hypothesis, we transferred L-se- Ϫ Ϫ ϩ ϩ Ϫ Ϫ TGCCL21 mice is independent of LT␣ lectin / and L-selectin / splenocytes into TGCCL21/RAG / mice and examined the thyroids 5 days later (n ϭ 5 mice/group). Genetic studies in mice have shown that members of the TNF/ As shown in Fig. 6, L-selectinϪ/Ϫ lymphocytes were found in the lymphotoxin family are important for lymphoid organ develop- thyroid in a pattern indistinguishable from that observed for the ment (49). Furthermore, expression of LT␣ has been documented L-selectinϩ/ϩ lymphocytes. To further analyze whether L-selectin in AITD (26). However, no information exists to date on the role played a major role in migration of cells to the thyroid we quan- of the lymphotoxin system in the generation of lymphoid aggre- tified the infiltrates by FACS. The number of CD45ϩ cells found gates in the thyroid. To test the hypothesis that the formation of in the thyroid of TGCCL21/RAGϪ/Ϫ mice after transfer of L-se- lymphoid aggregates induced by CCL21 requires LT␣, we crossed Ϫ Ϫ lectinϩ/ϩ and L-selectinϪ/Ϫ splenocytes was similar (average the TGCCL21 with LT␣ / mice to generate mice deficient in LT␣, expressing CCL21 in the thyroid (TGCCL21/LT␣Ϫ/Ϫ). When examined at 6 wk of age TGCCL21/LT␣Ϫ/Ϫ mice (n ϭ 8) and LT␣Ϫ/Ϫ mice (n ϭ 3) had no peripheral lymph nodes. How- ever, TGCCL21/LT␣Ϫ/Ϫ, but not LT␣Ϫ/Ϫ mice, had thyroid mononuclear infiltrates composed of B and T cells that were or- ganized in distinct areas (Fig. 7B) similar to that observed in the TGCCL21/LT␣ϩ/ϩ mice (Fig. 7A). In addition, PNAd-positive HEVs were found within both TGCCL21/LT␣ϩ/ϩ (Fig. 7C) and TGCCL21/LT␣Ϫ/Ϫ (Fig. 7D) thyroids. Thus, LT␣ is not required for accumulation of T, B cells, or the formation of HEVs induced by CCL21 in the thyroid. FIGURE 6. L-selectinϪ/Ϫ T cells can enter the thyroid of TGCCL21/ RAGϪ/Ϫ mice. Immunohistological analysis of the thyroid of TGCCL21/ Discussion RAGϪ/Ϫ mice transferred with 107 L-selectinϩ/ϩ (A) and L-selectinϪ/Ϫ (B) Despite the existence of solid experimental data linking human splenocytes. Thyroids were collected 5 days after cell transfer and stained thyroid disease and expression of chemokines, the studies to date with anti-CD3 Ab. Original magnification: AÐB, ϫ100. have been mostly descriptive. Little is know about the mechanisms 4796 TRANSGENIC EXPRESSION OF CCL21 IN THE THYROID

which lack expression of the L-selectin ligand PNAd, it under- scores a major difference between the thyroid gland and peripheral lymph nodes in which the recruitment of naive lymphocytes ␣ largely depends on L-selectin. Whether 4 integrins and their li- gands, which can mediate rolling interactions similar to the selec- tins (56), contribute to lymphocyte recruitment is a likely possi- bility that will be tested in future studies. We also show that the main chemokine receptor mediating CCL21 activity in the thyroid is CCR7 because mice that lack CCR7 expression did not exhibit thyroid infiltrates. Our results suggest that expression of the other murine CCL21 receptors (CXCR3 and CCX-CKR) is not sufficient to promote lymphocyte migration to the thyroid. However, whether these receptors have any role in migration of lymphocytes to the thyroid during inflam- matory conditions remains to be tested. Given the central role of LT␣ in lymphoid tissue development (49, 59–61), we wondered whether LT␣ was required for CCL21- induced lymphoid neogenesis. We have evaluated this hypothesis

FIGURE 7. Lymphocyte infiltration in the thyroid of TGCCL21 ani- by crossing TGCCL21 mice with mice deficient in LT␣ (35). In- Downloaded from mals is independent of LT␣. Cell infiltrates in the thyroid of TGCCL21/ terestingly, mice deficient in LT␣, expressing CCL21 in the thy- ϩ ϩ Ϫ Ϫ LT␣ / (A) and TGCCL21/LT␣ / mice (B). Notice the presence and roid (TGCCL21/LT␣Ϫ/Ϫ) had mononuclear infiltrates with sepa- topological segregation of T cells (CD3ϩ, green) and B cells (B220ϩ, red). ϩ ϩ rate B and T cells areas and HEV formation that was ␣ / ϩ ϩ PNAd-positive HEVs were present in both TGCCL21/LT (C) and indistinguishable from TGCCL21/LT␣ / mice. Similarly, mice ␣Ϫ/Ϫ ϫ TGCCL21/LT (D) thyroids. Original magnification: AÐB, 100; deficient in LT␣, expressing CCL21 in the pancreas also had in- CÐD, ϫ200.

filtrates with clear segregation of T and B cells (our unpublished http://www.jimmunol.org/ results). Finally, treatment of RIP-CCL21 mice with LT␤R-Fc af- fected the expression of PNAd and mucosal addressin cell adhe- or the nature of the factors regulating the influx and the fate of the sion molecule-1 on HEV but had a minor effect in reducing the cells infiltrating the thyroid gland. In the present report we tested cellularity of pancreatic infiltrates induced by CCL21 (29). In light the hypothesis that expression of CCL21 is sufficient to promote of these combined results, we suggest that LT␣-independent path- leukocyte entry into the thyroid. CCL21 is expressed in autoim- ways control development and organization of specific lymphoid mune thyroid disease (26) and has been implicated in the devel- aggregates induced by CCL21. The existence of LT␣-independent opment of ectopic lymphoid tissues in this organ (20, 26, 50). We pathways has been invoked to explain development of nasopha- show that expression of CCL21 in the thyroid of transgenic mice ryngeal-associated lymphoid tissue (62, 63). Initiation of nasopha- by guest on September 25, 2021 promotes recruitment of T and B lymphocytes to the thyroid. Most ryngeal-associated lymphoid tissue development happens mostly of the cells are naive, and are arranged in discrete compartments, after birth and, similar to what was observed here, it depends on resembling those observed in lymph nodes. LT␣ and L-selectin. Surprisingly, ectopic lymphoid neogenesis in- In lymphoid tissue, trafficking of naive T and B cells is regulated duced by another homeostatic chemokine (CXCL13) clearly re- by chemokines acting at the level of specialized endothelial struc- quires LT␣ (64). Our results raise the intriguing possibility that the tures known as HEV. Chemokines such as CCL21 (51, 52) and requirements for induction of ectopic lymphoid neogenesis in- CXCL13 (53) are expressed in the HEVs of lymph nodes and duced by these two chemokines may involve different signaling Peyer’s patches and trigger the activation of integrins present in pathways. lymphocytes, a process that depends on L-selectin and CCR7 (54, Although it is indisputable that lymphocytes play a role in the 55). Because our histological analysis showed the presence of pathogenesis of AITD, the function of the lymph node-like struc- HEVs within the thyroid of TGCCL21 mice, we initially asked tures often found in the thyroid in AITD remains unresolved. It has whether these structures were directly induced by CCL21 and es- been proposed that the presence of lymphoid follicles in areas rich sential for the influx of lymphocytes into the thyroid. Our studies in Ag may represent a site for magnification of the immune re- showed that HEVs are not formed within the thyroid of TGCCL21 sponse (20). We observed the presence of organized lymphoid mice that lack T and B cells (TGCCL21/RAGϪ/Ϫ mice), indicat- aggregates in the thyroid of TGCCL21 mice that resemble those ing that CCL21 expressed in the thyroid does not directly induce found in the thyroids of patients with AITD. Despite this similar- the differentiation of thyroid endothelial cells. Our studies also ity, none of the transgenic mice examined (n Ͼ 50) developed show that adoptively transferred lymphocytes infiltrate the signs of thyroid dysfunction (growth abnormalities and infertility) TGCCL21/RAGϪ/Ϫ thyroids, suggesting that infiltration of lym- within the first year of life. Serum thyroid hormone levels, as well phocytes is independent of HEV formation. As shown here, 60 as levels of TSH (which are elevated in hypothyroidism), did not days after transfer, vessels expressing PNAd are found within the differ between control and transgenic mice (data not shown). We large clusters of adoptively transferred cells. These results suggest also screened for TG autoantibodies in the serum of nontreated that HEV formation may depend on the infiltrating cells and, thus, transgenic (n ϭ 15) and wild-type littermates (n ϭ 12) (ages 65– may be a later event on the formation of the lymphoid aggregates. 381 days), and found no differences between these groups. Thus, The exact contribution of the incoming cells to the formation of the the presence of lymphocytes organized into follicular structures HEVs remains to be determined. within the thyroid was not sufficient to initiate autoimmunity in Unexpectedly, we found that the initial recruitment of lympho- TGCCL21 mice generated in the B6D2 background (MHC cyte in CCL21 expressing thyroid was L-selectin independent. Al- H-2b,d). Failure to develop autoimmunity may be due to multiple though this finding is consistent with the observation that lympho- causes, including lack of proper lymphocyte activation and/or ac- cytes can infiltrate the thyroids of TGCCL21/RAGϪ/Ϫ mice, tive lymphocyte suppression. Studies to examine the susceptibility The Journal of Immunology 4797 of the TGCCL21 mice to autoimmunity and their response to con- 8. Farber, J. M. 1997. Mig and IP-10: CXC chemokines that target lymphocytes. ventional immunization will require backcrossing of the TG- J. Leukocyte Biol. 61:246. 9. Romagnani, P., M. Rotondi, E. Lazzeri, L. Lasagni, M. Francalanci, GCCL21 mice into susceptible genetic backgrounds. A. Buonamano, S. Milani, P. Vitti, L. Chiovato, M. Tonacchera, et al. 2002. The mechanisms whereby CCL21 induces lymphoid neogenesis Expression of IP-10/CXCL10 and MIG/CXCL9 in the thyroid and increased levels of IP-10/CXCL10 in the serum of patients with recent-onset Graves’ dis- are not known, but appear to be context dependent. When over- ease. Am. J. Pathol. 161:195. expressed in the skin (27) or brain (30), CCL21 does not promote 10. Bjorkdahl, O., K. A. Barber, S. J. Brett, M. G. Daly, C. Plumpton, lymphocyte recruitment and organization. However, when ex- N. A. Elshourbagy, J. P. Tite, and L. L. Thomsen. 2003. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues. pressed in the thyroid or pancreatic islets (27–29), CCL21 induces Immunology 110:170. development of infiltrates rich in T and B cells, which segregate 11. Constantin, G., M. Majeed, C. Giagulli, L. Piccio, J. Y. Kim, E. C. Butcher, and ␤ into separate compartments, with T cells at the center of the infil- C. Laudanna. 2000. Chemokines trigger immediate 2 integrin affinity and mo- ϩ bility changes: differential regulation and roles in lymphocyte arrest under flow. trates. These infiltrates also contain CD11c dendritic cells and Immunity 13:759. ϩ very small numbers of macrophages (F4/80 cells) (27). The rea- 12. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and son for this apparent tissue specificity is not clear, and may include M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23. processing of CCL21, presence of CCL21-responding cells that 13. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster. 2000. could promote presentation or processing of CCL21 in the endo- Coexpression of the chemokines ELC and SLC by T zone stromal cells and thelium of “susceptible tissues”, or the responsiveness of the local deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97:12694. endothelium. 14. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and The development of the models described here (in particular, the H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ che- Ϫ Ϫ TGCCL21/RAG / mice) should now facilitate the characterization mokine have defects in lymphocyte homing and dendritic cell localization. Downloaded from J. Exp. Med. 189:451. of the mechanisms used by CCL21 to promote lymphocyte entry into 15. Muller, G., U. E. Hopken, H. Stein, and M. Lipp. 2002. Systemic immunoregu- nonlymphoid tissue. For instance, it will now be possible to define the latory and pathogenic functions of homeostatic chemokine receptors. J. Leuko- phenotype and developmental properties of the cells initially recruited cyte Biol. 72:1. ϩ Ϫ ϩ ϩ 16. Aust, G., M. Steinert, S. Kiessling, M. Kamprad, and C. Simchen. 2001. Reduced into the thyroid. Fetal lymphoid CD4 CD3 ROR␥t IL-7r␣ cells, expression of stromal-derived factor 1 in autonomous thyroid adenomas and its also known as lymphoid tissue inducer cells have been implicated in regulation in thyroid-derived cells. J. Clin. Endocrinol. Metab. 86:3368. 17. Aust, G., M. Steinert, C. Boltze, S. Kiessling, and C. Simchen. 2001. GRO-␣ in the development of lymphoid structures during embryogenesis (65, http://www.jimmunol.org/ normal and pathological thyroid tissues and its regulation in thyroid-derived cells. 66). Cells with similar properties may exist at reduced numbers in J. Endocrinol. 170:513. adult mice, or may be produced in specificinflammatory settings. The 18. Garcia-Lopez, M. A., D. Sancho, F. Sanchez-Madrid, and M. Marazuela. 2001. Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 model described here will allow us to define if lymphoid tissue in- ϩ and Mig and attract CXCR3 lymphocytes. J. Clin. Endocrinol. Metab. 86:5008. ducer cells are functionally relevant for the CCL21-induced formation 19. Kemp, E. H., R. A. Metcalfe, K. A. Smith, M. N. Woodroofe, P. F. Watson, and of ectopic lymphoid tissue in adult mice. A. P. Weetman. 2003. Detection and localization of chemokine gene expression In summary, we have shown that CCL21, a chemokine ex- in autoimmune thyroid disease. Clin. Endocrinol. 59:207. 20. Armengol, M. P., M. Juan, A. Lucas-Martin, M. T. Fernandez-Figueras, pressed in AITD, is sufficient to drive naive T and B cell recruit- D. Jaraquemada, T. Gallart, and R. Pujol-Borrell. 2001. Thyroid autoimmune ment into the thyroid. This process is mediated by CCR7 and re- disease: demonstration of thyroid antigen-specific B cells and recombination-

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