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* Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029;
Schering-Plough Research Institute, Kenilworth, NJ 07033;
DNAX Research, Palo Alto, CA 94304; and
Max-Delbruck-Center for Molecular Medicine, Berlin, Germany
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Abstract |
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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 in the thyroid. |
Introduction |
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1.5% of the general population (1). The major forms of AITD are Hashimoto’s thyroiditis (HT), causing hypothyroidism, and Graves’ disease (GD), causing hyperthyroidism. The hallmark of these conditions is thyroid dysfunction associated with the presence of lymphocytic infiltrates in the thyroid (2, 3). In HT, there is a diffuse lymphocytic infiltration with the presence of macrophages and destroyed thyrocytes. The lymphocytes often cluster to form lymphoid follicles with germinal centers. Lymphocytic infiltrates, usually without germinal center formation, are also seen in GD (4), but diffuse follicular cell hyperplasia and increased vascularity, rather than thyrocyte destruction, are observed (3). The etiopathogenic role of lymphocytes in HT and GD has been amply studied and a variety of effector mechanisms have been defined for both T and B cells (2, 5). T cells cause thyroid destruction directly, via cytotoxicity, or indirectly, through cytokines that induce apoptosis of thyroid follicle cells (5). B cells produce autoantibodies directed against thyroid Ags such as thyroglobulin (TG), thyroid peroxidase, and the thyroid-stimulating hormone (TSH) receptor (TSH-R) (2). These autoantibodies can either stimulate or inhibit thyroid function.
Despite substantial clinical and experimental data supporting a role for lymphocytes in triggering and sustaining AITD, factors responsible for their recruitment and retention in the thyroid remain uncertain. Migration of T cells appears to be regulated by inflammatory (inducible) and constitutive chemokines. Examples of inflammatory chemokines include CXCL8, CCL2, CCL3, CCL4, and CXCL10. Expression of these chemokines occurs during inflammation and is required for attraction of specific leukocyte subsets mediating inflammatory reactions (6, 7). CXCL10, the best studied of the inflammatory chemokines regulating T cell migration, is induced by IFN-
, a key cytokine involved in inflammation and immune regulation (8). CXCL10 interacts with CXCR3, a chemokine receptor expressed by effector T cells and endothelial cells (9).
Chemokines constitutively produced by lymphoid organs (CCL19, CCL21, CXCL12, and CXCL13) are involved in physiological trafficking of leukocytes and their segregation into specialized compartments. Among these, CCL19 and CCL21 are the best-characterized chemokines regulating homeostatic T cell migration. Both chemokines interact with a common receptor, CCR7, which is expressed by naive and memory T cells (10). CCL21 and CCL19 are displayed by high endothelial venules (HEVs) and activate integrins after interaction with CCR7 present on circulating lymphocytes (11). The functional consequence of integrin activation is increased adhesiveness of lymphocytes to the endothelium, a key step in lymphocyte transendothelial migration. Accordingly, mice lacking CCR7, CCL19, and CCL21 have defective homing of T cells into lymphoid tissue (12, 13, 14). In addition to controlling the initial aspects of lymphocyte migration, chemokines also control targeted homing of T and B cells within lymphoid organs. The specific segregation of B and T cells in secondary lymphoid organs is dependent on multiple chemokine gradients established by dendritic cells, stromal cells, and lymphocytes (15).
Chemokines, with the exception of CXCL12 (16), are not expressed by the normal human thyroid at appreciable levels. However, most chemokines studied to date are expressed within the thyroids of GD and HT patients. These include CXC chemokines, CXCL1 (17), the IFN- inducible chemokines CXCL9, and CXCL10 (9, 18, 19), CXCL12 (16), and CXCL13 (20). The CC chemokines CCL2, CCL3, CCL4, and CCL5 are also reported to be expressed in thyroid disease (19, 21, 22). Thyroid expression of these chemokines may be critical for the recruitment of chemokine receptor-expressing leukocytes to the thyroid. Indeed, CXCR3, CCR2, and CCR5 expression is elevated on infiltrating inflammatory cells relative to peripheral blood leukocytes (9, 18, 21, 23). The cellular source of these chemokines in the thyroid is usually the infiltrating lymphocytes, but thyrocytes and fibroblasts also express chemokines in diseased thyroid (17). Different proinflammatory stimuli such as IL-1, IFN-, and TNF- increase expression of chemokines by thyroid follicular cells in culture (17, 18, 24, 25), suggesting that inflammatory cytokines may trigger expression of chemokines by thyrocytes and other resident thyroid cells in vivo. Interestingly, recent studies have shown that chemokines required for the formation and maintenance of lymphoid follicles (i.e., CXCL12, CCL21, and CXCL13) are expressed in the thyroids of AITD patients (20, 26). The expression of these molecules in the thyroid suggests that chemokines may be necessary for recruitment and subsequent organization of lymphoid follicles in the thyroid. CCL21 and CXCL13 have been shown to induce formation of lymphoid structures when expressed in the pancreas (27, 28, 29). However, this property does not seem to be general, as expression of CCL21 in brain and skin of transgenic mice fails to induce development of such structures (30). Here, we directly test the hypothesis that the chemokine CCL21 may be directly implicated in regulating influx of lymphocytes into the thyroid. We show that CCL21 is sufficient to drive not only influx, but also formation of lymph node-like structures in the thyroid of transgenic animals, suggesting that this chemokine and its receptor, CCR7, may be involved in the formation of lymphoid follicles in the thyroid of patients with AITD. Additionally, we show that infiltration of the thyroid by lymphocytes under these conditions is independent of L-selectin and lymphotoxin (LT).
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Materials and Methods |
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The rat TG promoter (GenBank X06162) was amplified by PCR from rat genomic DNA using recognition primers that contained sites for HindIII at the 5' end and for EcoRI at the 3' end to include nucleotides –868 to –1 from the transcription start site. The rat TG promoter was subcloned into HindIII/EcoRI sites of pBS plasmid (Stratagene, La Jolla, CA) to generate the TG/pBS plasmid.
A BstX I fragment (2.4 kb) containing CCL21 genomic DNA encoding CCL21a (6Ckine serine) was isolated from a BAC clone (31) and blunt-end ligated into the EcoRI site of the TG/pBS plasmid to generate a vector encoding the TGCCL21 transgene. The TGCCL21 transgene (3.3 kb) was released from the TG/pBS vector by digestion with XhoI/XbaI restriction enzymes. Separation of the transgene DNA from vector DNA was accomplished by zonal sucrose gradient centrifugation as described (32). The 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 mM EDTA).
Mice
The TGCCL21 transgene DNA was resuspended in microinjection buffer to a final concentration of 1–5 ng/µl, microinjected into fertilized eggs from (C57BL/6J x DBA/2)F2 females (The Jackson Laboratory, Bar Harbor, ME), and transferred into oviducts of ICR foster mothers (Charles River Laboratories, Wilmington, MA) according to published procedures (33). Genotyping of the TGCCL21 transgenic founders and progeny was conducted by PCR analysis of mouse tail DNA using recognition primers for the rat TG promoter 5'-CTG CAG ACA AGC AGG CAT GCA-3' (forward) and 5'-CAC ACA TGG CAC ATA TGC-3' (reverse) as previously described (34). The endogenous low density lipoprotein gene (LDL) was used as an internal control using primers LDLL 5'-CGC AGT GCT CCT CAT CTG ACT TGT-3' (forward) and LDLU 5'-ACC CCA AGA CGT GCT CCC AGG ATG A-3' (reverse). PCR conditions were 94°C, 30 s; 60°C, 30 s; 72°C, 60 s for 30 cycles.
TGCCL21 transgenic mice (line 24) were crossed to CCR7–/– mice, described by Forster et al. (12), to LT–/– (35) and to RAG-2–/– mice (36). Mice hemizygous for CCL21 and null for RAG or CCR7 or LT (referred to as TGCCL21/RAG–/– mice, TGCCL21/CCR7–/– mice, and TGCCL21/LT–/– mice, respectively) and littermate controls were used for the experiments described here. Transgenic mice expressing GFP (37) and L-selectin–/– mice (38) were backcrossed over 10 generations in the C57BL/6 background. All mice were housed under specific-pathogen-free conditions in individually ventilated cages at the Mount Sinai School of Medicine Animal Facility. All experiments were performed following institutional guidelines.
Histology
Tissues for light microscopic examination were fixed by immersion in 10% phosphate-buffered formalin and then processed for paraffin sections. Routinely, 5-µm sections were cut and stained with H&E. For immunohistochemical staining, fresh frozen sections were first fixed with ice-cold acetone for 20 min, dried, and stored at –20°C. Slides were stained and analyzed as described previously (39). Slides were incubated for 1 h at room temperature with purified primary Abs followed by incubation with 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), peripheral lymph node addressin (PNAd) (MECA79) from BD Biosciences (San Diego, CA), and anti-6Ckine/CCL21 (no. AF457) from R&D Systems (Minneapolis, MN). Secondary Abs used were Alexa Fluor 594 donkey 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 Molecular Probes (Eugene, OR) and FITC and rhodamine red-X goat anti-Armenian hamster (nos. 127-095-160 and 127-295-160) and Cy5 goat anti-rat (no. 112-175-167) from Jackson ImmunoResearch Laboratories (West Grove, PA).
Flow cytometry
To prepare single cell suspension, individual thyroids from TGCCL21 animals (6 wk of age) were minced in serum-free RPMI 1640 media containing 0.0012 U/ml dispase I (Sigma-Aldrich, St. Louis, MO) and 0.25 U/ml collagenase type II (Worthington Biochemical/Invitrogen Life Technologies, Rockville, MD), incubated for 2 h at 4°C followed by an additional incubation of 10 min at 37°C. Thyroid cell suspensions obtained from both lobes were centrifuged at 250 x g for 10 min at 4°C and resuspended in FACS staining buffer (PBS containing 2% FCS and 0.01% sodium azide).
Cells were incubated for 20 min at 4°C with 5 µg/ml Fc block (BD Pharmingen, San Diego, CA) and then stained with directly conjugated primary mAbs. mAbs to the following mouse leukocyte surface markers were purchased from BD Pharmingen: CD45 (30-F11), CD3e (145-2C11), CD4 (RM4-5), B220 (RA3-6B2), CD62L (MEL-14), CD11c (HL-3), and CD11b (M1/70). To determine viability, samples were subsequently stained with 20 µl of 5 µg/ml propidium iodide (Calbiochem, La Jolla, CA). Events were acquired on a BD Biosciences FACScan and analyzed using CellQuest software. To rule out the presence of rare thymic contaminants in the thyroid single cell preparations, we routinely determined the relative proportion of thymocytes or immature T cells (CD4+/CD8+ cells). Samples that contained >2% of this cell population were excluded from the analysis.
Adoptive cell transfers
Spleen cells (107) were resuspended in PBS and injected i.v. Lymph nodes and thyroids from recipient animals were collected at various points (24, 48, 72, and 96 h) and fixed in 1.5% paraformaldehyde containing 20% sucrose for 24 h at 4°C. Fixed tissues were frozen in OCT compound (Tissue Tek, Torrance, CA), cryosectioned (8 mm), and examined under a fluorescent microscope. In some instances, images were overlaid and processed using Adobe Photoshop.
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Results |
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CCL21 is expressed in the thyroid of patients with AITD (20, 26), but it is unclear whether it has any role in pathogenesis. To investigate 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 constructed a transgene (TGCCL21) in which the CCL21 gene was placed downstream of a segment of the rat thyroglobulin promoter (–868 to –1 bp from the transcription start site). This promoter directs the expression of transgenes specifically to thyroid follicular cells (40, 41, 42, 43, 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 immunohistochemical 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 observed in other transgenic experiments (30). No immunoreactivity for CCL21 was observed in the control thyroids (Fig. 1A).
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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 = 5) (Fig. 1C). However, mononuclear infiltrates were observed in the thyroid of 5 of 8 founders examined (ages 74–208 days old). The infiltrates were composed of mononuclear cells and varied in size from mild (<20% involvement) to severe (>50% involvement). Analysis of thyroid sections from transgenic mice in line 24 (16–250 days old) also showed mononuclear infiltrates (Fig. 1D) in most (13 of 17) of the animals analyzed. Immunohistochemical staining using Abs against cell surface markers showed that both T and B lymphocytes were present in the cellular infiltrates (Fig. 1E) and appeared to be organized into separate clusters throughout the thyroid infiltrates (Fig. 1F). Occasionally, germinal center-like structures were observed in the thyroid of aged mice (>300 days), but no significant mitotic activity was observed within these structures (not shown). Scattered throughout the transgenic thyroid, we observed HEVs (Fig. 1G) expressing PNAd (Fig. 1H), an Ag marking the presence of L-selectin ligands in lymph nodes. Taken together, these results indicate that expression of CCL21 within the thyroid promotes accumulation of T and B cells and development of HEVs.
Thyroid infiltrates are composed of naive T and B cells
Histological analysis of the thyroid of TGCCL21 animals (6 wk of age) revealed a moderate mononuclear cell infiltration (20–50% of the thyroid occupied by mononuclear cells). To further characterize the composition of these infiltrates, we performed flow cytometry analyses using Abs against several leukocyte cell surface markers. Single cell suspensions of thyroids from control and transgenic mice were prepared according to the method described by Caturegli et al. (45). A small fraction of CD45+ cells were found in the control thyroids (<2% of total cells, n = 4). In contrast, CD45+ cells represented 30–50% of all cells in the thyroid of TGCCL21 animals at 6 wk of age (n = 10). The CD45+ cells were mostly (70%) T cells (CD3+), the majority of which were CD4+ T cells (Fig. 2). B cells (B220+) accounted for 27% of the infiltrates and the remaining 3% of the cells were dendritic cells. Most of the T cells expressed high levels of L-selectin (CD62L), suggesting a naive phenotype.
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Expression of CCL21 is a major factor regulating recruitment of naive and memory T cells into lymphoid tissues (46). Although there are at least three known receptors in mice for CCL21 (CCR7, CXCR3, and an unclassified receptor referred to as CCX-CKR (47, 48)), recent data suggest that the main receptor mediating CCL21-induced lymphocyte recruitment into lymphoid organs is CCR7 (46). To examine whether CCR7 mediates the recruitment of lymphocytes into the thyroid, we bred CCR7–/– mice with TGCCL21 mice to generate animals that expressed CCL21 in the thyroid, but lacked CCR7 expression (TGCCL21/CCR7–/– mice). Histological examination of thyroids from TGCCL21/CCR7+/+ or TGCCL21/CCR7+/– mice (n = 10 mice/group) revealed infiltration by mononuclear cells (Fig. 3A). In contrast, none of the thyroids from TGCCL21/CCR7–/– mice (n = 10) examined had leukocytic infiltrates when examined by light (Fig. 3B) or immunofluorescence microscopy using Abs against the pan-leukocyte marker CD45 (not shown). These findings indicate that CCR7 is the main chemokine receptor mediating CCL21-induced lymphocyte recruitment into the thyroid.
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HEVs, specialized blood vessels used by circulating lymphocytes to migrate into lymphoid organs, were present in the thyroid parenchyma of TGCCL21 mice. Next, we asked whether CCL21 could directly promote the development of HEVs in this tissue. To this end, we crossed the TGCCL21 mice with RAG-2–/– mice, which are devoid of mature T and B cells and examined the presence of HEVs in the thyroid (Fig. 4). As expected, significant infiltrates were observed in the thyroid of TGCCL21/RAG+/+ mice (n = 5) whereas no infiltrates were observed in the thyroid of TGCCL21/RAG–/– mice (n = 8), by H&E staining (Fig. 4, A and B, respectively) or CD45 staining (not shown). HEVs expressing PNAd (Fig. 4C), were found within TGCCL21/RAG+/+ thyroids. In contrast, no HEVs were observed in the TGCCL21/RAG–/– thyroid (Fig. 4D), suggesting that CCL21 is not sufficient to drive HEV formation and that factors produced by the infiltrating lymphocytes and/or dendritic cells may be important for the development of these structures in the tissue.
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To test whether CCL21 expression could promote recruitment of donor lymphocytes to the thyroid, we transferred 107 GFP+ splenocytes (i.v.) into the TGCCL21/RAG–/– animals described above (n = 8). GFP+ splenocytes were also injected into control RAG–/– mice (n = 5) that did not express CCL21 in the thyroid (Fig. 5B). Five days later we examined the presence of GFP+ cells in frozen sections of lymph nodes, spleen, and thyroids of TGCCL21/RAG–/– and RAG–/– mice. As expected, GFP+ cells accumulated in lymph nodes (Fig. 5A, a and b) and spleens (not shown) of both TGCCL21/RAG–/– and RAG–/– mice. Interestingly, clusters of GFP+ cells were visualized in the thyroid of the TGCCL21/RAG–/– mice (Fig. 5C), but not in the thyroid of RAG–/– mice (Fig. 5B) 5 days after transfer. Most of the infiltrating GFP+ cells at this time point were CD4+ T cells (not shown). Sixty days after transfer, large clusters of GFP+ cells were observed in the thyroid (Fig. 5D) (n = 2). These cells were mostly T and B cells that appeared to segregate into specific areas (Fig. 5E). Within these clusters we observed PNAd+ vessels (Fig. 5F). These results indicate that expression of CCL21 by thyroid follicular cells promotes mobilization of adoptively transferred lymphocytes into the thyroid parenchyma.
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The absence of both lymphoid infiltrates and HEVs in TGCCL21/RAG–/– suggested that the presence of HEVs was important for lymphocytic recruitment. To test this possibility, we transferred donor wild-type splenocytes into TGCCL21/RAG–/– mice and examined thyroid specimens for the presence of HEVs and expression of PNAd. We found no HEV-like structures, or PNAd expression up to 96 h after adoptive transfer, indicating that HEVs are not required for initial colonization of the thyroid by lymphocytes (data not shown).
The absence of PNAd expression suggested that the initial lymphocyte infiltration might have occurred through an L-selectin-independent pathway. To test this hypothesis, we transferred L-selectin–/– and L-selectin+/+ splenocytes into TGCCL21/RAG–/– mice and examined the thyroids 5 days later (n = 5 mice/group). As shown in Fig. 6, L-selectin–/– lymphocytes were found in the thyroid in a pattern indistinguishable from that observed for the L-selectin+/+ lymphocytes. To further analyze whether L-selectin played a major role in migration of cells to the thyroid we quantified the infiltrates by FACS. The number of CD45+ cells found in the thyroid of TGCCL21/RAG–/– mice after transfer of L-selectin+/+ and L-selectin–/– splenocytes was similar (average 2.8 x 103 cells vs 2.2 x 103 cells, n = 3). These results indicate that L-selectin is not required for initial entry of lymphocytes into the thyroid of TGCCL21/RAG–/– mice.
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Genetic studies in mice have shown that members of the TNF/lymphotoxin family are important for lymphoid organ development (49). Furthermore, expression of LT has been documented in AITD (26). However, no information exists to date on the role of the lymphotoxin system in the generation of lymphoid aggregates in the thyroid. To test the hypothesis that the formation of lymphoid aggregates induced by CCL21 requires LT, we crossed the TGCCL21 with LT–/– mice to generate mice deficient in LT, expressing CCL21 in the thyroid (TGCCL21/LT–/–). When examined at 6 wk of age TGCCL21/LT–/– mice (n = 8) and LT–/– mice (n = 3) had no peripheral lymph nodes. However, TGCCL21/LT–/–, but not LT–/– mice, had thyroid mononuclear infiltrates composed of B and T cells that were organized in distinct areas (Fig. 7B) similar to that observed in the TGCCL21/LT+/+ mice (Fig. 7A). In addition, PNAd-positive HEVs were found within both TGCCL21/LT+/+ (Fig. 7C) and TGCCL21/LT–/– (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.
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Discussion |
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In lymphoid tissue, trafficking of naive T and B cells is regulated by chemokines acting at the level of specialized endothelial structures known as HEV. Chemokines such as CCL21 (51, 52) and CXCL13 (53) are expressed in the HEVs of lymph nodes and Peyer’s patches and trigger the activation of integrins present in lymphocytes, a process that depends on L-selectin and CCR7 (54, 55). Because our histological analysis showed the presence of HEVs within the thyroid of TGCCL21 mice, we initially asked whether these structures were directly induced by CCL21 and essential for the influx of lymphocytes into the thyroid. Our studies showed that HEVs are not formed within the thyroid of TGCCL21 mice that lack T and B cells (TGCCL21/RAG–/– mice), indicating that CCL21 expressed in the thyroid does not directly induce the differentiation of thyroid endothelial cells. Our studies also show that adoptively transferred lymphocytes infiltrate the TGCCL21/RAG–/– thyroids, suggesting that infiltration of lymphocytes is independent of HEV formation. As shown here, 60 days after transfer, vessels expressing PNAd are found within the large clusters of adoptively transferred cells. These results suggest that HEV formation may depend on the infiltrating cells and, thus, may be a later event on the formation of the lymphoid aggregates. The exact contribution of the incoming cells to the formation of the HEVs remains to be determined.
Unexpectedly, we found that the initial recruitment of lymphocyte in CCL21 expressing thyroid was L-selectin independent. Although this finding is consistent with the observation that lymphocytes can infiltrate the thyroids of TGCCL21/RAG–/– mice, which lack expression of the L-selectin ligand PNAd, it underscores 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 ligands, which can mediate rolling interactions similar to the selectins (56), contribute to lymphocyte recruitment is a likely possibility 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 inflammatory conditions remains to be tested.
Given the central role of LT in lymphoid tissue development (49, 59, 60, 61), we wondered whether LT was required for CCL21-induced lymphoid neogenesis. We have evaluated this hypothesis by crossing TGCCL21 mice with mice deficient in LT (35). Interestingly, mice deficient in LT, expressing CCL21 in the thyroid (TGCCL21/LT–/–) had mononuclear infiltrates with separate B and T cells areas and HEV formation that was indistinguishable from TGCCL21/LT+/+ mice. Similarly, mice deficient in LT, expressing CCL21 in the pancreas also had infiltrates with clear segregation of T and B cells (our unpublished results). Finally, treatment of RIP-CCL21 mice with LT
R-Fc affected the expression of PNAd and mucosal addressin cell adhesion molecule-1 on HEV but had a minor effect in reducing the cellularity of pancreatic infiltrates induced by CCL21 (29). In light of these combined results, we suggest that LT-independent pathways control development and organization of specific lymphoid aggregates induced by CCL21. The existence of LT-independent pathways has been invoked to explain development of nasopharyngeal-associated lymphoid tissue (62, 63). Initiation of nasopharyngeal-associated lymphoid tissue development happens mostly after birth and, similar to what was observed here, it depends on LT and L-selectin. Surprisingly, ectopic lymphoid neogenesis induced by another homeostatic chemokine (CXCL13) clearly requires LT (64). Our results raise the intriguing possibility that the requirements for induction of ectopic lymphoid neogenesis induced by these two chemokines may involve different signaling pathways.
Although it is indisputable that lymphocytes play a role in the pathogenesis of AITD, the function of the lymph node-like structures often found in the thyroid in AITD remains unresolved. It has been proposed that the presence of lymphoid follicles in areas rich in Ag may represent a site for magnification of the immune response (20). We observed the presence of organized lymphoid aggregates in the thyroid of TGCCL21 mice that resemble those found in the thyroids of patients with AITD. Despite this similarity, none of the transgenic mice examined (n > 50) developed signs of thyroid dysfunction (growth abnormalities and infertility) within the first year of life. Serum thyroid hormone levels, as well as levels of TSH (which are elevated in hypothyroidism), did not differ between control and transgenic mice (data not shown). We also screened for TG autoantibodies in the serum of nontreated transgenic (n = 15) and wild-type littermates (n = 12) (ages 65–381 days), and found no differences between these groups. Thus, the presence of lymphocytes organized into follicular structures within the thyroid was not sufficient to initiate autoimmunity in TGCCL21 mice generated in the B6D2 background (MHC H-2b,d). Failure to develop autoimmunity may be due to multiple causes, including lack of proper lymphocyte activation and/or active lymphocyte suppression. Studies to examine the susceptibility of the TGCCL21 mice to autoimmunity and their response to conventional immunization will require backcrossing of the TGGCCL21 mice into susceptible genetic backgrounds.
The mechanisms whereby CCL21 induces lymphoid neogenesis are not known, but appear to be context dependent. When overexpressed in the skin (27) or brain (30), CCL21 does not promote lymphocyte recruitment and organization. However, when expressed in the thyroid or pancreatic islets (27, 28, 29), CCL21 induces development of infiltrates rich in T and B cells, which segregate into separate compartments, with T cells at the center of the infiltrates. These infiltrates also contain CD11c+ dendritic cells and very small numbers of macrophages (F4/80+ cells) (27). The reason for this apparent tissue specificity is not clear, and may include processing of CCL21, presence of CCL21-responding cells that could promote presentation or processing of CCL21 in the endothelium of "susceptible tissues", or the responsiveness of the local endothelium.
The development of the models described here (in particular, the TGCCL21/RAG–/– mice) should now facilitate the characterization of the mechanisms used by CCL21 to promote lymphocyte entry into nonlymphoid tissue. For instance, it will now be possible to define the phenotype and developmental properties of the cells initially recruited into the thyroid. Fetal lymphoid CD4+CD3–RORt+IL-7r+ cells, also known as lymphoid tissue inducer cells have been implicated in the development of lymphoid structures during embryogenesis (65, 66). Cells with similar properties may exist at reduced numbers in adult mice, or may be produced in specific inflammatory settings. The model described here will allow us to define if lymphoid tissue inducer cells are functionally relevant for the CCL21-induced formation of ectopic lymphoid tissue in adult mice.
In summary, we have shown that CCL21, a chemokine expressed in AITD, is sufficient to drive naive T and B cell recruitment into the thyroid. This process is mediated by CCR7 and results in accumulation and topological arrangement of lymphocytes within the thyroid. Adoptive transfer experiments indicate that the initial recruitment of lymphocytes is independent of L-selectin and the presence of HEVs. Once the cells infiltrate the adult thyroid, aggregates are formed with segregation of T and B cells into specific compartments, resembling the ectopic, lymph node-like structures observed in many autoimmune and chronic inflammatory conditions, including AITD. The generation of the genetic models described here will facilitate the analysis of the mechanisms leading to the formation of these lymphoid structures in adult animals and to the definition of their role in disease.
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Acknowledgments |
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Footnotes |
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1 S.A.L. is an Irene Diamond Associate Professor of Immunology. This work was supported in part by a grant from the Irene Diamond Fund, and by Grants DK 067989 (to S.A.L.) and HL 69438 (to P.S.F.) from the National Institutes of Health. 
2 A.P.M., E.C.C., and G.C.F. contributed equally to the manuscript.
3 Address correspondence and reprint requests to Dr. Sergio A. Lira, Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1630, New York, NY, 10029-6574. E-mail address: sergio.lira{at}mssm.edu
4 Abbreviations used in this paper: AITD, autoimmune thyroid diseases; HT, Hashimoto’s thyroiditis; GD, Graves’ disease; TG, thyroglobulin; TSH, thyroid-stimulating hormone; HEV, high endothelial venule; Lt, lymphotoxin ; PNAd, peripheral lymph node addressin; LDL, low density lipoprotein.
Received for publication June 4, 2004. Accepted for publication July 30, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13:759.[Medline]
in normal and pathological thyroid tissues and its regulation in thyroid-derived cells. J. Endocrinol. 170:513.[Abstract]
and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue. Eur. J. Immunol. 27:2600.[Medline]
in the thyroid. Proc. Natl. Acad. Sci. USA 97:1719./mCCL19: comparison to human CCX-CKR. Eur. J. Immunol. 32:1230.[Medline]
4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers. Immunol. Rev. 156:137.[Medline]
(LT ) and retinoic acid receptor-related orphan receptor-, but the organization of NALT is LT dependent. J. Immunol. 168:986.R, and NIK signaling pathways but requires the Id2 gene and CD3–CD4+CD45+ cells. Immunity 17:31.[Medline]
t in the development of lymph nodes and Peyer’s patches. Immunol. Rev. 195:81.[Medline]
(t) in the generation of fetal lymphoid tissue inducer cells. Nat. Immunol. 5:64.[Medline]Related articles in The JI:
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 H. Unsoeld, K. Mueller, U. Schleicher, C. Bogdan, J. Zwirner, D. Voehringer, and H. Pircher Abrogation of CCL21 chemokine function by transgenic over-expression impairs T cell immunity to local infections Int. Immunol., November 1, 2007; 19(11): 1281 - 1289. [Abstract] [Full Text] [PDF]
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 J. Rangel-Moreno, J. E. Moyron-Quiroz, L. Hartson, K. Kusser, and T. D. Randall Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local immunity to influenza PNAS, June 19, 2007; 104(25): 10577 - 10582. [Abstract] [Full Text] [PDF]
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 A. P. Martin, J. M. Alexander-Brett, C. Canasto-Chibuque, A. Garin, J. S. Bromberg, D. H. Fremont, and S. A. Lira The Chemokine Binding Protein M3 Prevents Diabetes Induced by Multiple Low Doses of Streptozotocin J. Immunol., April 1, 2007; 178(7): 4623 - 4631. [Abstract] [Full Text] [PDF]
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G. C. Furtado, T. Marinkovic, A. P. Martin, A. Garin, B. Hoch, W. Hubner, B. K. Chen, E. Genden, M. Skobe, and S. A. Lira Lymphotoxin beta receptor signaling is required for inflammatory lymphangiogenesis in the thyroid PNAS, March 20, 2007; 104(12): 5026 - 5031. [Abstract] [Full Text] [PDF]
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R. Le Panse, G. Cizeron-Clairac, J. Bismuth, and S. Berrih-Aknin Microarrays Reveal Distinct Gene Signatures in the Thymus of Seropositive and Seronegative Myasthenia Gravis Patients and the Role of CC Chemokine Ligand 21 in Thymic Hyperplasia J. Immunol., December 1, 2006; 177(11): 7868 - 7879. [Abstract] [Full Text] [PDF]
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 A. Antonelli, M. Rotondi, S. M. Ferrari, P. Fallahi, P. Romagnani, S. S. Franceschini, M. Serio, and E. Ferrannini Interferon-{gamma}-Inducible {alpha}-Chemokine CXCL10 Involvement in Graves' Ophthalmopathy: Modulation by Peroxisome Proliferator-Activated Receptor-{gamma} Agonists J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 614 - 620. [Abstract] [Full Text] [PDF]
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P. N. Pichurin, C.-R. Chen, G. D. Chazenbalk, H. Aliesky, N. Pham, B. Rapoport, and S. M. McLachlan Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization J. Immunol., January 1, 2006; 176(1): 668 - 676. [Abstract] [Full Text] [PDF]
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 K. Chen, Y. Wei, A. Alter, G. C. Sharp, and H. Braley-Mullen Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., September 1, 2005; 78(3): 716 - 724. [Abstract] [Full Text] [PDF]
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