LTβR signaling induces cytokine expression and upregulates lymphangiogenic factors in lymph node anlagen

The formation of lymph nodes is a complex process crucially controlled through triggering of LTβR on mesenchymal cells by LTα1β2 expressing lymphoid tissue inducer (LTi) cells. This leads to the induction of chemokines to attract more hematopoietic cells and adhesion molecules to retain them. Here we show that the extravasation of the first hematopoietic cells at future lymph node locations occurs independently of LTα and that these cells, expressing TRANCE, are the earliest LTi cells. By paracrine signalling the first expression of LTα1β2 is induced. Subsequent LTβR triggering on mesenchymal cells leads to their differentiation to stromal organizers, which now also start to express TRANCE, Il7 as well as VEGF-C, in addition to the induced adhesion molecules and chemokines. Both TRANCE and Il7 will further induce the expression of LTα1β2 on newly arrived immature LTi cells, resulting in more LTβR triggering, generating a positive feedback loop. Thus, LTβR triggering by LTi cells during lymph node development creates a local environment to which hematopoietic precursors are attracted and where they locally differentiate into fully mature, LTα1β2 expressing, LTi cells. Furthermore, the same signals may regulate lymphangiogenesis to the lymph node through induction of VEGF-C. 55 LTβR signaling induces cytokine expression and upregulates lymphangiogenic factors in lymph node anlagen Introduction The development of lymph nodes (LNs) depends on the cross talk between hematopoietic cells and mesenchymal stromal cells 1 6, whereby the inductive signal for lymph node development comes from CD4+Il7r+CD3-CD45+RORγ(t)+ hematopoietic cells. These lymphoid tissue inducer cells (LTi cells) express lymphotoxin-α1β2 (LTα1β2) and are thought to trigger lymphotoxin β receptor (LTβR) expressed on stromal cells 2, 7, 8. The importance of the LTβR signaling lies in the induction of adhesion molecules and production of chemokines that start a set of events leading to the formation of the lymph node. Initially, LTβR ligation leads to activation of the classical NF-κB pathway, which results in the expression of proinflammatory molecules such as the chemokines MIP1β, MIP-2, and the adhesion molecule VCAM-1. Prolonged triggering results in the activation of the alternative NF-κB pathway and the generation of p52, involved in the transcription of the lymphoid chemokines such as CCL21, CCL19 and CXCL13 9, 10. These chemokines in particular are crucial for the development of the lymph node 2, 11 15. The expression of LTα1β2 itself can equally well be induced by signaling through the interleukin-7 receptor (Il7r) as well as the tumor necrosis factor–related activationinduced cytokine-receptor (TRANCE-R; also known as Rank, Tnfrsf11a, ODFR or Ly109) upon binding of the respective ligands, Il7 or TRANCE 3. For lymph node development it was shown that signaling through both receptors is critical, while during Peyer’s patch formation only Il7r triggering is mandatory 2, 3, 5, 15 17. Although TRANCE has been reported to be expressed by LTi as well as stromal organizer cells in developing lymph nodes, Il7 was shown to be expressed in stromal organizer cells of developing Peyer’s patches 8, 18, 19. The importance of limited Il7r triggering for lymphoid organ development can be deduced from experiments in which transgenic overexpression of Il7 resulted in an increased number of LTi cells, leading to a 5 fold increase in the number of Peyer’s patches and the formation of ectopic lymphoid structures 20. In contrast, transgenic overexpression of TRANCE did not affect the number of LTi cells on a wild type background, while it was able to rescue the reduced number of LTi cells in developing lymph nodes of TRANCE-/mice 18. It is unclear how expression of TRANCE and Il7 is regulated during lymph node development, although analysis of the TRANCE promoter region revealed response elements for Vitamin D3 and glucocorticoids 21, 22, as well as binding sites for Runx2 and NF-κB 23, while LTβR signaling has been proposed to result in enhanced Il7 production 20. However, it has been reported that initial clustering of LTi cells can occur in lymphotoxin alpha (LTα) -/mice, and therefore other mechanisms precede the crucial triggering of LTβR on stromal organizer cells 24, 25. In this study, we show that the expression of TRANCE on LTi cells is unaffected in LN anlagen from LTα -/mice, but that LTα1β2-mediated LTβR signaling is essential for the expression of TRANCE on stromal cells. The earliest phase of lymph node development is marked by the clustering of LTi cells, which occurs independent of LTα1β2-LTβR, followed by the LTα1β2-mediated differentiation of stromal cells. The LTα1β2-induced differentiation of stromal cells leads to the upregulation of VCAM-1,