Lymphotoxin beta receptor signaling is required for inflammatory lymphangiogenesis in the thyroid

Abstract

Infiltration of lymphocytes into the thyroid gland and formation of lymph node-like structures is a hallmark of Hashimoto's thyroiditis. Here we demonstrate that lymphatic vessels are present within these infiltrates. Mice overexpressing the chemokine CCL21 in the thyroid (TGCCL21 mice) developed similar lymphoid infiltrates and lymphatic vessels. TGCCL21 mice lacking mature T and B cells (RAGTGCCL21 mice) did not have cellular infiltrates or increased number of lymphatic vessels compared with controls. Transfer of CD3(+)CD4(+) T cells into RAGTGCCL21 mice promoted the development of LYVE-1(+)podoplanin(+)Prox-1(+) vessels in the thyroid. Genetic deletion of lymphotoxin beta receptor or lymphotoxin alpha abrogated development of lymphatic vessels in the inflamed areas in the thyroid but did not affect development of neighboring lymphatics. These results define a model for the study of inflammatory lymphangiogenesis in the thyroid and implicate lymphotoxin beta receptor signaling in this process.

Fig. 1.

Lymphatic vessels are present within lymphoid aggregates in Hashimoto's thyroiditis. (A) H&E-stained thyroid sections from patients with Hashimoto's thyroiditis showed the presence of the mononuclear cells aggregates. (B) Lymphocytic infiltration was visualized by using anti-CD3 and anti-CD20 antibodies. (C) Podoplanin-positive lymphatic vessels were present within lymphocytic infiltrates (visualized by CD3 staining) in the thyroids of all cases studied (n = 4). (D and E) Higher magnification of lymphatic vessels stained with podoplanin (D) and Prox-1 (E) antibodies. (F) Colocalization of podoplanin and Prox-1 in lymphatic vessels. (Scale bars, 25 μm.)

Fig. 2.

Lymphatic vessels are present within lymphoid aggregates in the thyroids of TGCCL21 mice. (A and B) Thyroids from wild-type (A) and TGCCL21 (B) mice were examined for the presence of lymphatic vessels. (B) LYVE-1⁺ vessels (magenta) were abundant in infiltrated areas of TGCCL21 thyroid (revealed by DAPI counterstain). (C) LYVE-1⁺ vessels (green) were present within both T cell (CD3, red) and B cell (B220, blue) areas. (D and E) LYVE-1⁺ vessels present in the thyroid of TGCCL21 mice (n = 6) also were positive for the endothelial marker CD31 (D) and lymphatic marker Prox-1 (arrowheads point to PROX-1⁺ nuclei) (E). (F) Confocal analysis of the whole-mount thyroid revealed the presence of a complex lymphatic network in the thyroid of TGCCL21 mice. (G–I) Flow-cytometric analysis revealed increased relative and absolute numbers of LYVE-1⁺CD31^low cells in the thyroid of TGCCL21 mice. Data are mean ± SEM (n = 4 mice; ∗∗, P < 0.01). (Scale bars, 25 μm.)

Fig. 3.

CD3⁺CD4⁺ T cells induce formation of lymphatic vessels in the thyroid. (A and B) Thyroids from RAG (A) and RAGTGCCL21 (B) mice were stained with anti-LYVE-1, anti-CD45 antibodies, and DAPI (blue). No obvious differences were observed between the two strains (n = 3 per group). (C) Splenocytes (10⁷) were injected i.v. into RAGTGCCL21 mice. Large numbers of CD45⁺ cells and LYVE-1⁺ vessels were observed in the thyroids 2 months after transfer (n = 3). (D) Purified CD3⁺CD4⁺ T cells (10⁶) were injected into RAGTGCCL21 mice. Ten days after transfer, LYVE-1⁺ vessels were detected in the thyroids of all RAGTGCCL21 mice examined (n = 5). (Scale bars, 100 μm.) (E) Relative levels of mRNA for the indicated genes in the thyroids of RAG, RAGTGCCL21, and RAGTGCCL21 mice injected with 10⁶ CD3⁺CD4⁺ T cells for 10 days were determined by quantitative PCR. Results are mean ± SEM. Three animals of each genotype were used in these experiments (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).

Fig. 4.

LTβR and LTα deficiency impairs formation of lymphatic vessels in the thyroid. (A) Flow-cytometric analysis of LTβR expression in LYVE-1, CD31, CD34, and CD45 cell subsets present in the thyroid of wild-type (n = 7) and TGCCL21 mice. The isotype control is represented by the gray tracing. Representative results of two independent experiments are shown. (B) Thyroids from TGCCL21, TGCCL21/LTβR^−/−, and TGCCL21/LTα^−/− mice were stained with anti-LYVE-1 and CD45 antibodies. Compared with TGCCL21 mice (n = 3), the number and size of LYVE-1⁺ vessels were significantly reduced in both TGCCL21/LTβR^−/− (n = 7) and TGCCL21/LTα^−/− (n = 3) mice. Representative staining is shown. (Scale bars, 250 μm.)