Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation

Abstract

The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue-inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5-14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons.

Figure 1. Specific migration of LTi cells and precursors towards chemokines

(a) Averaged specific migration values of cell populations from E12.5–14.5 (n = 8 independent experiments with each containing at least 8 embryos). CD4⁺CD45⁺ represent LTi cells. Error bars in the graph represent the standard error. (b) Representative FACS analysis (of 8 independent experiments) of hematopoietic cells that responded to the indicated chemokine. Cells were additionally stained for c-Kit and CD4, allowing identification of LTi precursor cells (c-Kit⁺CD4⁻) and LTi cells (c-Kit⁺CD4⁺).

Figure 2. Chemokines CXCL13 and CCL21 are present in E13.5 developing lymph nodes

Embryonic LN anlagen (n = 8) were first identified by Madcam1 and CD4 localization. (a) The cervical LN anlagen was stained for chemokines CCL21 (arrow in a) and CXCL13 in a subsequent section (b). (c) A highly magnified area of the cervical LN shown in (a–b) stained for CXCL13. (a) To identify hematopoietic cells within the anlagen, CD45 was used. (a–c). To identify stromal organizer and lymphatic endothelial cells, Podoplanin was used. (c–e) Lymphatic endothelial cells are identified as Lyve-1⁺ and Podoplanin^high, while stromal organizer cells are Podoplanin⁺ and VCAM1⁺ (b). The white lines represent blood vessels (a–b). (d–e) Also other LN anlagen, such as the axillary or more posterior located iliac LN, were stained for CXCL13, Madcam1, and Lyve-1. (f) The subsequent section of the iliac LN anlagen, was stained for Lyve-1, Ccl21, and CD4. Bars represent 25 µm in c and 75 µm in others.

Figure 3. LN development and CXCL13 expression occur independent of LTβR signaling

(a) mRNA levels of CXCL13 were measured in E12.5–E13 Lta^−/− and wild type embryonic mesenchymal cells (n = 4). (b) Embryonic LN anlagen from E14.5 Lta^−/− were stained for CXCL13 (arrow), CD4, and Podoplanin. Bar represents 75 µm.

Figure 4. Retinoic acid induces the expression of chemokine CXCL13 via RARβ

(a) CXCL13, CCL21, VCAM1, and Madcam1 mRNA in E13.5 Bl/6 embryonic cells in the presence of RA for 6h compared to untreated cells (n = 5). (b) CXCL13 mRNA in unstimulated mesenchymal cells from E12.5–E13.5 Lta^−/− and Bl/6 wild type embryos (n =7) and after RA treatment. (c) RARβ or (d) CXCL13 mRNA in unstimulated mesenchymal cells from E13.5 Bl/6 wild type embryos and after RA treatment (n = 5), in the presence (+LE) or absence (vehicle) of specific RARβ inhibitors LE540 and LE135 (LE). (e) CXCL13 or RARβ mRNA in unstimulated mesenchymal cells from E13.5 Bl/6 wild type embryos, after RA treatment (n = 8), and after RA treatment in the presence of cycloheximide (Chx). Error bars represent the standard error. NS means not statistically significant (P > 0.05). (f–g) Cervical LN anlagen in E14.5 DR5-luciferase embryo (n = 4) stained for (f) Madcam1, CD4, CD45, or (g) podoplanin, Cxcl13, and luciferase (arrowheads). Bars represent 75 µm.

Figure 5. Most LN anlagen are lacking in Raldh2^−/− E14.5 embryos

In Raldh2^−/− embryos at E14.5 (n = 3), serial sections of LN anlagen in the cervical area were stained for CCL21, CD4 and Madcam1 (a), and CXCL13, CD45 and VCAM1 (b). (c) Anti-Lyve-1 was used to stain lymphatic endothelial cells. (d) LTi cells were stained with anti-CD45, anti-CD4 and anti-RORγt (arrowheads in d). Bars represent 75 µm.

Figure 6. Raldh2 is localized in neurons adjacent to LN anlagen

E13.5 embryos (n = 8) were stained for Raldh1 and 2 in combination with CXCL13 (a–c) and neuronal marker anti-βIII-Tubulin (d). Shown are cervical (a) or peripheral thymic (b-detail in c) LN anlagen in which LTi cells are identified by IL7Rα. Bars represent 75 µm.

Figure 7. Stimulated neurons lead to CXCL13 expression

The right cervical vagal nerve was electrically stimulated (VNS) in adult mice and CXCL13 expression was analyzed in the ileum. As controls, animals were sham operated in which the vagal nerve was exposed, but not electrically stimulated (sham). (a) CXCL13 mRNA in the ileum at 4, 8, and 24 hrs after VNS (n = 3 independent experiments for each bar). (b) CXCL13 mRNA in the ileum at 8 hrs after VNS in the presence of injected RARβ blockers LE135 and LE540 (VNS LE) or vehicle (VNS ETOH) and in sham operated animals that received LE135 and LE1540 (sham LE) or vehicle alone (sham ETOH). (n = 6–9 independent experiments. Error bars represent the standard error.