Regulation of lymph node vascular growth by dendritic cells

Lymph nodes grow rapidly and robustly at the initiation of an immune response, and this growth is accompanied by growth of the blood vessels. Although the vessels are critical for supplying nutrients and for controlling cell trafficking, the regulation of lymph node vascular growth is not well understood. We show that lymph node endothelial cells begin to proliferate within 2 d of immunization and undergo a corresponding expansion in cell numbers. Endothelial cell proliferation is dependent on CD11c+ dendritic cells (DCs), and the subcutaneous injection of DCs is sufficient to trigger endothelial cell proliferation and growth. Lymph node endothelial cell proliferation is dependent on vascular endothelial growth factor (VEGF), and DCs are associated with increased lymph node VEGF levels. DC-induced endothelial cell proliferation and increased VEGF levels are mediated by DC-induced recruitment of blood-borne cells. Vascular growth in the draining lymph node includes the growth of high endothelial venule endothelial cells and is functionally associated with increased cell entry into the lymph node. Collectively, our results suggest a scenario whereby endothelial cell expansion in the draining lymph node is induced by DCs as part of a program that optimizes the microenvironment for the ensuing immune response

A Dendritic-Cell-Stromal Axis Maintains Immune Responses in Lymph Nodes

SummaryWithin secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTβR) ligands were critical mediators, and LTβR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTβR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases

Normalization of the Lymph Node T Cell Stromal Microenvironment in lpr/lpr Mice Is Associated with SU5416-Induced Reduction in Autoantibodies

The vascular-stromal elements of lymph nodes can play important roles in regulating the activities of the lymphocytes within. During model immune responses, the vascular-stromal compartment has been shown to undergo proliferative expansion and functional alterations. The state of the vascular-stromal compartment and the potential importance of this compartment in a spontaneous, chronic model of autoimmunity have not been well studied. Here, we characterize the vascular expansion in MRL-lpr/lpr lymph nodes and attempt to ask whether inhibiting this expansion can interfere with autoantibody generation. We show that characteristics of vascular expansion in enlarging MRL-lpr/lpr lymph nodes resemble that of the VEGF-dependent expansion that occurs in wild-type mice after model immunization. Surprisingly, treatment with SU5416, an inhibitor of VEGF and other receptor tyrosine kinases, did not have sustained effects in inhibiting vascular growth, but attenuated the anti-dsDNA response and altered the phenotype of the double negative T cells that are expanded in these mice. In examining for anatomic correlates of these immunologic changes, we found that the double negative T cells are localized within ectopic follicles around a central B cell patch and that these T cell-rich areas lack the T zone stromal protein ER-TR7 as well as other elements of a normal T zone microenvironment. SU5416 treatment disrupted these follicles and normalized the association between T zone microenvironmental elements and T cell-rich areas. Recent studies have shown a regulatory role for T zone stromal elements. Thus, our findings of the association of anti-dsDNA responses, double negative T cell phenotype, and altered lymphocyte microenvironment suggest the possibility that lymphocyte localization in ectopic follicles protects them from regulation by T zone stromal elements and functions to maintain autoimmune responses. Potentially, altering the lymphocyte microenvironment that is set up by the vascular-stromal compartment can be a means by which to control undesired autoimmune responses

Optical projection tomography reveals dynamics of HEV growth after immunization with protein plus CFA and features shared with HEVs in acute autoinflammatory lymphadenopathy

The vascular-stromal compartment of lymph nodes is important for lymph node function, and high endothelial venules (HEVs) play a critical role in controlling the entry of recirculating lymphocytes. In autoimmune and autoinflammatory diseases, lymph node swelling is often accompanied by apparent HEV expansion and, potentially, targeting HEV expansion could be used therapeutically to limit autoimmunity. In previous studies using mostly flow cytometry analysis, we defined three differentially regulated phases of lymph node vascular-stromal growth: initiation, expansion, and the re-establishment of vascular quiescence and stabilization. In this study, we use optical projection tomography to better understand the morphologic aspects of HEV growth upon immunization with ovalbumin/CFA (OVA/CFA). We find HEV elongation as well as modest arborization during the initiation phase, increased arborization during the expansion phase, and, finally, vessel narrowing during the re-establishment of vascular quiescence and stabilization. We also examine acutely enlarged autoinflammatory lymph nodes induced by regulatory T cell depletion and show that HEVs are expanded and morphologically similar to the expanded HEVs in OVA/CFA-stimulated lymph nodes. These results reinforce the idea of differentially regulated, distinct phases of vascular-stromal growth after immunization and suggest that insights gained from studying immunization-induced lymph node vascular growth may help to understand how the lymph node vascular-stromal compartment could be therapeutically targeted in autoimmune and autoinflammatory diseases

SU5416 reduces anti-dsDNA and alters T cell phenotype but without sustained effect on vascular growth.

<p>(A–D) MRL-lpr/lpr mice (JAX 006825) were treated for either 2 weeks (A) or 11.5 weeks (B–D) with indicated treatments starting at 8 weeks of age. (E–G) MRL-lpr/lpr (JAX 000485) were treated for 5–6 weeks with SU5416 starting at 10 weeks of age. (A) Endothelial cell numbers in brachial lymph nodes after 2 weeks of SU5416 treatment. n = 3 mice per condition. (B) Endothelial cell numbers in brachial lymph nodes after 11.5 weeks of SU5416 treatment. n = 4 DMSO mice and 3 SU5416 mice. Representative of 2 similar experiments. (C) Anti-dsDNA titers and (D) total IgG titers in serum over time. For (C–D), ELISA for serum from 0, 2, and 4 weeks was run independently from serum at 8, 10, and 11.5 weeks. n = 4 mice per treatment except at 11.5 weeks, when n = 3 for SU5416 (see text). Representative of 2 similar experiments. (E) Number of plasma cells per organ. n = 4 mice per treatment over 3 experiments. (F) Number of anti-dsDNA-secreting cells as determined by ELISPOT. n = 4 mice per treatment over 3 experiments. (G) Syndecan levels on indicated T cell populations from SU5416 or DMSO-treated mice. Representative of 4 mice over 3 experiments. For (A–F), * = p<.05 and * = p<.01 when SU5416 treatment compared to DMSO treatment using t-test.</p

Follicles of double negative T cells exclude T zone constituents and SU5416 normalizes the microenvironment.

<p>MRL-lpr-lpr mice were treated with SU5416 or DMSO vehicle for 11.5 weeks starting at 8 weeks of age and lymph nodes were examined. For comparison to a stimulated wild-type lymph node, 10 week old MRL+/+ mice were immunized with OVA/CFA and draining brachial lymph nodes were examined at day 8. (Part i of A–F), nearby sections from a single day 8 MRL+/+ brachial lymph node were stained as indicated. (Part ii of A–F), nearby sections from a single brachial lymph node from a DMSO-treated MRL-lpr/lpr mouse were stained as indicated. (Part iii of A–F), higher magnification of boxed area in corresponding images in part ii. (Part iv of A–F), nearby sections from a single brachial lymph node from a SU5416 -treated MRL-lpr/lpr mouse were stained as indicated. (Part v of A–F), higher magnification of boxed area in corresponding images in part iv. (G–H) Higher magnification of (G) medullary and (H) T zone areas in (Fi). “F” denotes follicle. (I–J) Higher magnification of (I) medullary-like and (J) T zone-like areas in (Fiii). (K) Higher magnification of normalized T zone area in (Fv). Scale bars represent 500 um.</p

MRL-lpr/lpr lymph node vasculature shows expansion and re-established quiescence with course of disease.

<p>MRL+/+ and MRL-lpr/lpr mice were examined at indicated ages; brachial lymph nodes were used for flow cytometry-based studies (A, C–E, G–J) and popliteal lymph nodes were used for VEGF determination (F). (A) Lymph node cellularity as determined by count of lymph node cells. (B) Serum anti-dsDNA IgG titers. (C) Number of endothelial cells per lymph node as determined by flow cytometry. “Total EC” = CD45^negCD31^pos cells; total endothelial cells are comprised of PNAd ^pos “HEV EC” and PNAd ^neg “nonHEV mixed EC.” (D) Endothelial cell proliferation rate as determined by the percent of endothelial cells that are BrdU+. (E) Number of proliferating (BrdU+) endothelial cells per lymph node. (F) VEGF levels in lymph nodes. Each symbol represents 1 mouse. (G) HEV trafficking efficiency. About 3×10⁷ CFSE-labeled splenocytes were intravenously injected into 3.5 month old mice at 30 minutes prior to sacrifice. HEV trafficking efficiency was defined as the number of CFSE-labeled splenocytes that entered the lymph node divided by the number of HEV endothelial cells. n = 3 mice per condition. Representative of 3 similar experiments. (H) Number of lymphocyte subsets over time. Cells were identified as follows: “B220+ T cells” were B220^posCD3^pos cells; “B220− T cells” were B220^negCD3^pos cells; “B cells” were B220^posCD3^neg cells. (I) Number of subsets of CD11c+ cells over time. (J) Ratio of CD11c^hi cells to CD11c^med cells over time. For A–E, H–J, n = 6 mice per condition over 3 experiments. For A–J, * = p<.05 and ** = p<.01 in comparison to the same measurement in the age matched MRL+/+ mice using t-test.</p

SU5416 induces infiltration of ER-TR7 into the B cell areas within ectopic follicles.

<p>MRL-lpr-lpr mice were treated with SU5416 or DMSO vehicle for 11.5 weeks starting at 8 weeks of age and lymph nodes were examined. For comparison to a stimulated wild-type lymph node, 10 week old MRL+/+ mice were immunized with OVA/CFA and draining brachial lymph nodes were examined at day 8. (A, D) nearby sections from a single brachial lymph node from a DMSO-treated MRL-lpr/lpr mouse were stained as indicated. (B, E) Nearby sections from a single axillary lymph node from a DMSO-treated MRL-lpr/lpr mouse were stained as indicated. (C,F) Nearby sections from a single axillary lymph node from a SU5416-treated MRL-lpr/lpr mouse were stained as indicated. (G) Higher magnification of B cell follicle border indicated in (D, arrow). (H) Higher magnification of ectopic follicle border indicated in (E, arrow). (I, J) Higher magnification of border of B cell areas indicated in (F). (I) is magnification of area denoted by single arrow and (J) is magnification of area denoted by double arrows. Scale bars represent 500 um.</p