Integrin-Alpha IIb Identifies Murine Lymph Node Lymphatic Endothelial Cells Responsive to RANKL

Microenvironment and activation signals likely imprint heterogeneity in the lymphatic endothelial cell (LEC) population. Particularly LECs of secondary lymphoid organs are exposed to different cell types and immune stimuli. However, our understanding of the nature of LEC activation signals and their cell source within the secondary lymphoid organ in the steady state remains incomplete. Here we show that integrin alpha 2b (ITGA2b), known to be carried by platelets, megakaryocytes and hematopoietic progenitors, is expressed by a lymph node subset of LECs, residing in medullary, cortical and subcapsular sinuses. In the subcapsular sinus, the floor but not the ceiling layer expresses the integrin, being excluded from ACKR4+LECs but overlapping with MAdCAM-1 expression. ITGA2b expression increases in response to immunization, raising the possibility that heterogeneous ITGA2b levels reflect variation in exposure to activation signals. We show that alterations of the level of receptor activator of NF-κB ligand (RANKL), by overexpression, neutralization or deletion from stromal marginal reticular cells, affected the proportion of ITGA2b+LECs. Lymph node LECs but not peripheral LECs express RANK. In addition, we found that lymphotoxin-β receptor signaling likewise regulated the proportion of ITGA2b+LECs. These findings demonstrate that stromal reticular cells activate LECs via RANKL and support the action of hematopoietic cell-derived lymphotoxin

ITGA2b⁺ LECs are responsive to RANKL and lymphotoxin activation.

<p>(A) Mean ± SD (n = 6) percentage of ITGA2b⁺ LECs of peripheral (p)LNs (inguinal, axial and brachial) versus mesenteric (mes) LNs. (B) Mice were immunized with heat-inactivated <i>B</i>. <i>pertussis</i> and LEC ITGA2b expression of draining and non-draining LNs was compared. The graph shows the mean ± SD (n = 6) percentage of ITGA2b⁺ LECs, revealing increased ITGA2b proportions in response to immunization. (C) Flow cytometry histograms display representative ITGA2b expression ±SD (n = 6) by stromal subsets of inguinal and popliteal LNs draining the immunization site. (D) Histograms show reactivity to anti-GPIBβ labelling of stromal subsets of inguinal and popliteal LNs draining the immunization site. The percentage ±SD (n = 6) of cells labelled by the antibody is indicated. (E) The increase in the proportion of ITGA2b⁺ pLN LECs from RANK-Tg mice (overproducing soluble RANKL in the skin) compared with LECs of WT controls is shown as mean ± SD (n = 8). (F) Graph shows reduction in the percentage of ITGA2b⁺ LECs upon RANKL neutralization (mean ± SD, n = 5). (G) Confocal microscopy imaging in inguinal LN subcapsular and medullary sinuses of ITGA2b expression (green) by LECs (10.1.1, magenta) after RANKL-neutralization or after administration of isotype-control antibody. (H) Mean ± SD (n = 9) percentage of ITGA2b⁺ LECs from mice with conditional deficiency of RANKL in marginal reticular cells (KO) versus WT littermate controls. (I) Histograms of ITGA2b expression of LECs from mice treated with LTβR-Ig or IgG1 control. The graph depicts the mean ± SD (n = 3) percentage of ITGA2b⁺ LECs. *p<0.5, **p < 0.01, ***p < 0.001.</p

ITGA2b is heterogeneously expressed in the adult and embryonic LN.

<p>(A) Confocal microscopy images of an adult inguinal LN probed for ITGA2b together with LEC marker mCLCA1 (mAb 10.1.1) in the subcapsular, the medullary and the cortical sinus. Scale bars are indicated. (B) Confocal microscopy images of an embryonic (E18.5) inguinal LN of ITGA2b and LEC marker Lyve-1. Higher magnification of boxed area is shown below. (C) Flow cytometry counterplot of ITGA2b versus ACKR4 expression by LN LECs of ACKR4-GFP transgenic mice. The percentage ±SD (n = 3) of positive cells is indicated. (D) Mean ± SEM <i>Itga2b</i> mRNA expression of total LN from mice aged 5 days and 8 weeks, and of MAdCAM-1⁺ and MAdCAM-1^- cell-sorted LECs from mice aged 8 weeks. Statistical analysis: ***p<0.001, ns = non significant by one way Anova with the Bonferroni method.</p