LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen

Abstract

The formation of lymph nodes is a complex process crucially controlled through triggering of LTbetaR on mesenchymal cells by LTalpha(1)beta(2) expressing lymphoid tissue inducer (LTi) cells. This leads to the induction of chemokines to attract more hematopoietic cells and adhesion molecules to retain them. In this study, we show that the extravasation of the first hematopoietic cells at future lymph node locations occurs independently of LTalpha and that these cells, expressing TNF-related activation-induced cytokine (TRANCE), are the earliest LTi cells. By paracrine signaling the first expression of LTalpha(1)beta(2) is induced. Subsequent LTbetaR triggering on mesenchymal cells leads to their differentiation to stromal organizers, which now also start to express TRANCE, IL-7, as well as VEGF-C, in addition to the induced adhesion molecules and chemokines. Both TRANCE and IL-7 will further induce the expression of LTalpha(1)beta(2) on newly arrived immature LTi cells, resulting in more LTbetaR triggering, generating a positive feedback loop. Thus, LTbetaR 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, LTalpha(1)beta(2) expressing, LTi cells. Furthermore, the same signals may regulate lymphangiogenesis to the lymph node through induction of VEGF-C.

FIGURE 1

E14.5 WT BLN anlage characterization. A, CD4⁺IL-7R⁺CD45^int and few immature CD4⁻IL-7R⁺CD45^int LTi cells are present in the E14.5 anlage (CD4 in green, IL-7R in red, and CD45 in blue). B, VCAM-1 expression (red) on stromal cells is limited at this time point, but ICAM-1 expression (blue) can be found on blood vessels and CD45⁺ hematopoietic cells (green). C, TRANCE expression (red) is limited to CD45⁺ hematopoietic cells (blue) of which a number are CD4⁺ LTi cells (green). Arrowhead points to TRANCE-expressing LTi cell (D) LTβR^int and MAdCAM-1⁺ stromal cells, LTβR⁺ endothelial cells, and CD4⁺ LTi cells (MAdCAM-1 in green, LTβR in red, CD4 in blue) occupy the E14.5 BLN anlage. E, MAdCAM-1⁺ stromal cells (green) colocalize with CD4⁺ LTi cells (blue) and TNF-R1⁺ cells (red). F, LN anlagen contain VEGFr2⁺MECA32^low and VEGFr2⁺MECA32⁺ blood vessels and are surrounded by VEGFR2⁺Lyve-1⁺ lymphatic endothelial cells (VEGFr2 in green, MECA32 in red, Lyve-1 in blue). A–F lenses used: ×40.

FIGURE 2

TRANCE expression in E14.5 WT Axillary LN and LTα^−/− rALN anlagen. A and B, ALN anlagen are found as clusters of hematopoietic cells in both WT and LTα^−/− at E14.5 (CD4 in green, IL-7R in red, and CD45 in blue). C and D, Subsequent sections were stained for MAdCAM-1 (green), TRANCE (red), and CD45 (blue). TRANCE expression is found on CD45⁺ hematopoietic cells in both WT (C) and LTα^−/− (D) LN anlagen. Arrowheads indicate MAdCAM-1⁺TRANCE⁺ stromal cells. At E14.5, MAdCAM-1⁺ cells surround TRANCE expressing cells in WT ALN anlagen and LTα^−/− rALN anlagen. A–D lenses used: ×40.

FIGURE 3

Altered morphology of E16.5 LTα^−/− BLN anlagen and lack of TRANCE expression on stromal cells. A, Clustered LTi cells are found in WT (A) BLN anlagen but not in LTα^−/− (B) BLN anlagen (CD4 in green, IL-7R in red, and CD45 in blue). At E16.5, large numbers of CD45⁺ hematopoietic cells (blue) and ICAM-1⁺VCAM-1⁺ stromal cells (ICAM-1 in green, VCAM-1 in red) are found in the WT BLN anlage (C) but not in the LTα^−/− BLN anlage (D). ICAM-1⁺VCAM-1^low vessels are found in both WT and LTα^−/− animals. TRANCE⁺VCAM-1⁺ and MAdCAM-1⁺ stromal cells are found in WT BLN anlage (E) but not in LTα^−/− BLN anlagen (TRANCE in green, VCAM-1 in red, MAdCAM-1 in blue) (G). BLN anlagen contain Lyve-1⁺VE-cadherin⁺ LECs, MAdCAM-1^int stromal cells, VE-cadherin⁺, and VE-cadherin⁺MAdCAM-1⁺ blood vessels (H). Both WT BLN anlagen (I) and LTα^−/− BLN anlagen (J) contain MECA32⁺VEGFr2⁺ blood vessels and a VEGFr1⁺VEGFr2⁺ blood vessel. In addition, VEGFr2⁺ lymphatics are present at this time point in development (VEGFr1 in green, MECA32 in red, VEGFr2 in blue). A–J lenses used: ×40.

FIGURE 4

LTβR triggering results in TRANCE up-regulation (A). Stimulation of LTβR with agonistic anti LTβR mAb for 8 h in LTα^−/− E18.5 rudimentary MLN cell cultures results in a significant increase in TRANCE mRNA expression compared with untreated cell cultures. Proper stimulation with the agonistic anti-LTβR mAb was validated by increase of VCAM-1 expression. Experiments were performed three times (B). Treatment of cultured WT MEFs with agonistic anti-LTβR mAb, but not with an isotype-matched control mAb, results in the up-regulation of TRANCE and VCAM-1 mRNA expression. MEFs were collected at 2, 4, 6, 24, and 30 h after stimulation for analysis of mRNA expression and (C) at 24 h after stimulation for analysis of ICAM-1, TRANCE, and VCAM-1 protein expression by FACS. Expression of transcripts in B was normalized to endogenous references genes as indicated. Relative expression levels at t = 0 were set at 1,0. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5

LTβR triggering leads to induction of IL-7 and VEGF-C. Treatment of cultured WT MEFs with agonistic LTβR mAb, but not with an isotype matched control mAb, results in the up-regulation of IL-7 (A) and VEGF-C (B) mRNA expression. MEFs were collected at 4, 6, 24, and 30 h after stimulation with agonistic LTβR mAb 4H8WH2. Expression of transcripts was normalized to endogenous references genes as indicated. Relative expression levels at t = 0 were set at 1,0. Experiments were performed three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001.