Growth by Stromal Cell Proliferation RANKL Induces Organized Lymph Node

RANKL Induces Organized Lymph Node Growth by Stromal Cell Proliferation Estelle Hess, Vincent Duheron, Marion Decossas, Frédéric Lézot, Ariane Berdal, Sylvestre Chea, Rachel Golub, Mattéo This information is current as R. Bosisio, S. Lori Bridal, Yongwon Choi, Hideo Yagita and of October 2, 2021. Christopher G. Mueller J Immunol 2012; 188:1245-1254; Prepublished online 30 December 2011; doi: 10.4049/jimmunol.1101513 http://www.jimmunol.org/content/188/3/1245 Downloaded from

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Material 3.DC1 http://www.jimmunol.org/ References This article cites 78 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/188/3/1245.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

RANKL Induces Organized Lymph Node Growth by Stromal Cell Proliferation

Estelle Hess,* Vincent Duheron,* Marion Decossas,* Fre´de´ric Le´zot,†,‡ Ariane Berdal,† Sylvestre Chea,x Rachel Golub,x Matte´o R. Bosisio,{,‖ S. Lori Bridal,{ Yongwon Choi,# Hideo Yagita,** and Christopher G. Mueller*

RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size,

inclusive area, and numbers of B cell follicles. Reticular ﬁbroblastic and vascular stromal cells, important for secondary lymphoid Downloaded from organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These ﬁndings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system. The Journal of Immunology, 2012, 188: 1245–1254.

he immune system displays a great capacity to adapt to response (1, 2). Stroma comprises vascular blood endothelial cells http://www.jimmunol.org/ environmental changes to provide the best-tailored re- (BECs), lymph endothelial cells (LECs), and fibroblastic reticular T sponse to a given immune challenge. Secondary lymphoid cells (FRCs), which are interconnected through a microconduit organs (SLOs), such as lymph nodes (LNs), play an important part system (3). The FRC population consists of reticular cells of the in this activity. They are the anatomical sites where information T cell zone (TRCs) and the sinus marginal zone (MRCs), as well from tissue is integrated by the immune cells. Over the last years, it as B cell follicular dendritic cells (FDCs) (4–7). Vascular cells has emerged that nonhematopoietic stromal cells that support the regulate entry and exit of lymphocytes (8–11), and FRCs function parenchyma of SLOs play a key role in orchestrating the immune in the homeostasis of T and B cells (12, 13); together, they provide the necessary structural framework for immune cell encounter and by guest on October 2, 2021 activation (2, 3, 12). During an immune response, stromal cells *Centre National de la Recherche Scientifique, Laboratoire d’Immunopathologie et Chimie The´rapeutique, Unite´ Propre de Recherche 9021, Institut de Biologie possess the plasticity to adapt to rapid SLO expansion with Molećulaire et Cellulaire, Universite´ de Strasbourg, 67084 Strasbourg, France; massive increases in lymphocyte numbers (7, 14). However, it is †INSERM, Unite´ Mixte de Recherche 872, Centre de Recherche des Cordeliers, Equipe 5, Laboratoire de Physiopathologie Orale et Molećulaire, Universite´ de not known how SLOs accomplish size increase and whether this Paris V-VI, 75006 Paris, France; ‡INSERM Unite´ Mixte de Recherche 957, Univer- process involves stroma. x site´ de Nantes, 44035 Nantes, France; Unite´ de Lymphopoie`se, INSERM U668, During embryogenesis, stroma interacts with lymphoid tissue Institut Pasteur and Universite´ de Paris Diderot, Sorbonne Cite´ (Cellule Pasteur), 75006 Paris, France; {Centre National de la Recherche Scientifique, Unite´ Mixte inducer (LTi) cells and orchestrates SLO development by pro- de Recherche 7623, Universite´ de Paris VI, 75006 Paris, France; ‖Echosens S.A., ducing chemokines and adhesion molecules (15–19). Stroma # Paris, France; Department of Pathology and Laboratory Medicine, University of expresses the lymphotoxin (LT) b receptor, which is an important Pennsylvania School of Medicine, Philadelphia, PA 19104; and **Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan molecule in this process (20, 21). LTbR and RANK are members Received for publication May 24, 2011. Accepted for publication November 30, of the TNFR superfamily (TNFRSF11a) and signal through the 2011. classical and the alternative NF-kB pathways (22, 23). RANK is This work was supported by Centre National de la Recherche Scientifique, University activated by RANKL (TNFSF11 or TRANCE), which is blocked of Strasbourg, and La Ligue Contre le Cancer (to E.H.). V.D. and E.H. were recip- by the soluble decoy receptor osteoprotegerin (OPG) (22, 24). ients of fellowships from Centre National de la Recherche Scientifique Bourse Doctorat Ingeńieur and Allocation Ministe´rielle de Recherche, respectively. RANK- and RANKL-deficient mice lack most LNs (25–27), Address correspondence and reprint requests to Dr. Christopher G. Mueller, Centre which renders the study of RANK function in LN immune ho- National de la Recherche Scientifique, Unite´ Propre de Recherche 9021, Universite´ meostasis difficult. LTi cells express RANK and RANKL, and de Strasbourg, 15 Rue Rene´ Descartes, 67084 Strasbourg, France. E-mail address: their numbers and cluster formation are reduced in RANKL [email protected] knockout (KO) mice (27). In addition, RANK stimulates LTa1b2 The online version of this article contains supplemental material. expression by LTi cells, the ligand for LTbR (28). This led to the Abbreviations used in this article: BEC, blood endothelial cell; BM, bone marrow; proposal that RANK-RANKL signaling in LTi cells results in cLN, cutaneous lymph node; DC, dendritic cell; FDC, B cell follicular dendritic cell; FRC, fibroblastic reticular cell; HF, hair follicle; iLN, inguinal lymph node; KLH, activation of LTbR on stromal cells and subsequent SLO forma- keyhole limpet hemocyanin; KO, knockout; LEC, lymph endothelial cell; LN, lymph tion. However, the rescue of LN organogenesis in Rankl2/2 mice node; LT, lymphotoxin; LTi, lymphoid tissue inducer; mLN, mesenteric lymph node; MRC, reticular cell of sinus marginal zone; NP, 4-hydroxy-3-nitrophenyl-acetyl; by transgenic RANKL expression in B or T cells does not restore OPG, osteoprotegerin; P, postnatal day; PTA, peripheral tissue Ag; qRT-PCR, quan- LTi cell numbers to normal levels, and the engagement of stromal titative RT-PCR; SLO, secondary lymphoid organ; Tg, transgenic; TRC, reticular cell cell LTbR by an agonistic Ab appears unable to normalize LN of T zone; WT, wild-type. formation (27). Moreover, Peyer’s patches and rescued mesenteric 2/2 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 LNs of Rankl mice are smaller (25, 27) and display disorga- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101513 1246 SECONDARY LYMPHOID ORGAN GROWTH BY RANKL nized B cell follicles (27, 29). Mice deficient in TRAF6, an taining 2% FCS (v/v; Lonza), Dispase (1 mg/ml; Roche), Collagenase D adaptor molecule of RANK signaling, lack LNs; LN formation (1 mg/ml; Roche), and DNase (40 mg/ml; Sigma-Aldrich). Medium can be rescued by exogenous IL-7 but the SLOs then lack clearly containing enzymes was then removed and renewed for another 20 min. The digest was gently pipetted every 10 min to break aggregates, until no defined B cell follicles (28). These findings underscore our in- visible fragment remained; the final pipetting was done in the presence of 5 complete knowledge of the prenatal and postnatal functions of mM EDTA. Cells were then passed through a 40-mm mesh and washed 2 RANK in SLO formation and homeostasis. twice in RPMI 1640 10% FCS (v/v). In some experiments, CD45 stromal The hair follicle (HF), one of the defining features of mammals, cells were isolated by negative selection using anti-CD45–coupled mag- netic microbeads (Miltenyi Biotec). Dendritic cells (DCs) were enriched is a miniorgan of the skin, which periodically regenerates by re- by an Optiprep gradient (Sigma-Aldrich) (41) after LN digestion with petitive cycles of growth (anagen), regression (catagen), and rel- Collagenase D (1 mg/ml) for 45 min under agitation. ative quiescence (telogen) (30). It has a specialized association with the immune system, because it enjoys a relative immune Flow cytometry and cell sorting privilege (31) and shares gene products with the thymus, most Single-cell suspensions obtained from different organs were stained with commonly exemplified by the Foxn-12/2 nude mice that lack hair Abs (Supplemental Table II) and analyzed on a FACSCalibur (Becton Dickinson) using CellQuest and FlowJo software. Stromal cells were sorted and thymus. RANK and RANKL are expressed in the thymus and 2 on a FACSVantage SE (Becton Dickinson) by gating on CD45 (allo- function in the formation of medullary thymic epithelial cells (32– phycocyanin) single cells and using podoplanin+ (FITC)- and CD31 (PE)- 36). RANK also plays an important role in HF biology, because specific Abs (purity was 95 6 2% for BECs, 95 6 3% for LECs, and 93 6 RANK stimulation of HF stem cells is required for entry into 2% for FRCs). Regulatory T cells were labeled for Foxp3 following anagen. Anagen HFs produce soluble RANKL (37). the manufacturer’s recommendations (eBioscience). Adult LTi-like cells, identified as lin2 (CD3, CD8, CD11c, TCRb, TCRgd, CD19, Ter119, In this article, we present an analysis of a mouse transgenic (Tg) + + +

NK1.1, Ly6C/G) RORgt a4b7 CD127 , were first incubated with an anti- Downloaded from for Rank under the control of the S100A8 promoter, active in HF CD16/CD32 Ab before being labeled for LTa1b2 using mouse LTbR fused stem cells (37) and in macrophages (38). The mouse displays to human IgG-Fc, followed by fluorochrome-conjugated goat anti-human a massive postnatal expansion of skin-draining LNs. The pro- IgG. To ensure specificity, a negative control sample was preincubated portions and organization of hematopoietic and stromal cell with anti-LTa and LTb mAbs before addition of mouse LTbR-human IgG-Fc. populations are well maintained, with the exception of an increase in small B cell follicles. The immune cells are not activated and Immunofluorescence respond rapidly to immunization. Using bone marrow (BM) trans- http://www.jimmunol.org/ LNs were frozen in Tissue-Tek O.C.T. Compound (Electron Microscopie fers, skin transplantation, and genetic rescue experiments, we Science), and sections were prepared. After fixation in cold acetone and showed that Tg Rank expression in HFs is responsible for LN blocking with goat serum (Sigma-Aldrich), they were incubated with growth. RANKL is overproduced from the Tg HFs and is re- primary Abs and, if necessary, secondary detection Abs (Supplemental sponsible for LN growth, because RANKL neutralization restores Table II). Images were acquired with a Zeiss Axiovert 200M microscope and AxioVision Rel.4.8 software. Quantification of area and number of normal organ size. The vascular and reticular fibroblastic cells B cell follicles was done on images using ImageJ software. b-galactosidase express RANK and undergo hyperproliferation, which is abro- activity was assessed, as described (42). gated by RANKL neutralization. Moreover, stromal cells show increased transcription of chemokines and cell-adhesion mole- RT-PCR by guest on October 2, 2021 cules, which would contribute to LN growth. Our data showed that RNA from sorted cells was extracted with RNeasy kits (Qiagen), and cDNA RANKL functions in SLO homeostasis by stimulating an equili- was synthesized with oligo(dT)15 primers and ImProm-II (Promega) or brated proliferation of all stromal cell subsets concomitant with Superscript III (Invitrogen). RT-PCR was performed using Promega GoTaq amplification mix and Eurogentec qPCR MasterMix Plus Low ROX. The increased lymphocyte recruitment. gene-specific primers are listed in Supplemental Table I. Quantitative RT-PCR (qRT-PCR) was run on a Stratagene MX4000 thermal cycler, Materials and Methods and threshold cycle values of target genes were normalized to GAPDH, Mice hypoxanthineguanine phosphoribosyltransferase, and b-actin. The expression factor was calculated using the Relative Expression Software Tool Animals were kept in a pathogen-free barrier facility, and experimentation (http://mmm.gene-quantification.de/rest.html). was performed in accordance with institutional guidelines and its animal bioethics accord (approval no. AL/01/20/11/08). All mutants were identified BrdU incorporation by PCR (Supplemental Table I), with the exception of CD45.1 mice, which Mice were injected i.p. with 0.5 mg BrdU (Sigma-Aldrich) diluted in PBS were phenotyped by flow cytometry for the CD45 isoform. Mice aged 6–8 wk 2 + were used for all experiments, unless stated otherwise. Ultrasound bio- and given BrdU in drinking water (0.8 mg/ml). CD45 and CD45 cells microscopy was performed with 24- and 47.8-MHz ultrasound systems on were isolated from LNs, and the incorporation of BrdU was measured by anesthetized mice (39). For adoptive BM transfer, 3-wk-old mice were le- flow cytometry using the BrdU flow kit (BD Pharmingen). Stromal subsets thally irradiated with 10-Gy x-rays (Particle accelerator, Saturn 41; General were identified using CD31 and podoplanin as markers. 6 Electric); 24 h later, they received 2 3 10 BM cells i.v., harvested from Immunization congenic CD45.1+ wild-type (WT) or CD45.2+ Tg mice. Chimerism was verified every month by assessing the percentage of donor blood leukocytes. Twelve-week-old mice received s.c. injections of 200 mg chicken OVA For skin transplantation, skin from age-matched mice was transplanted onto with 5 mg LPS (Sigma-Aldrich), IFA, or 25 mg CpG (InvivoGen). A boost the back, below the neck, of 3 wk-old nude mice. A healing spray was ap- was administrated 2 wk later. Before and every 5 d thereafter, chicken plied, and mice were killed 7 wk later. The Rank transgene was backcrossed OVA-reactive serum IgG was measured by direct ELISA. For Ab- onto Msx-2lacZ/lacZ CD1 mice for more than eight generations, which affinity measures, mice received s.c. injections of 50 mg 4-hydroxy-3- maintained cutaneous LN (cLN) hyperplasia, although it was less pro- nitrophenyl-acetyl (NP)30-keyhole limpet hemocyanin (KLH) (Biosearch nounced. LN cell numbers were determined at 3 mo of age. To block in vivo Technologies) in 5 mg LPS (Sigma-Aldrich). A boost was administrated RANK–RANKL interaction, the neutralizing anti-RANKL Ab IK22-5 (40) at 10 d. Serum anti-NP IgG was determined every 5 d by direct ELISA was injected s.c. into Rank-Tg mice every 3 d (10 mg at postnatal day [P] 10, on plates coated with NP3-BSA or NP30-BSA (Biosearch Technologies). 25 mg from P13 to P28, and 50 mg from P31 to P45). Mock-treated mice Titers were determined as highest serum dilution that gave a value of at received azide-free rat IgG (Jackson Immunochemicals). least three SD above the average reading for secondary controls. The relative affinity was determined as the titer NP /titer NP (43). Isolation of stromal cells and lymphocytes 3 30 Statistical analysis Stromal cells were isolated and identified using CD31 and podoplanin as markers, as described (12). Briefly, LNs were collected, and capsules were An unpaired two-tailed Student t test was used to determine statistically opened and incubated for 30 min at 37˚C in RPMI 1640 (Lonza) con- significant differences. The p values , 0.05 were considered statistically The Journal of Immunology 1247 significant. GraphPad Prism version 5 for Windows (GraphPad software) into follicles of the cortical area but the presence of small B cell was used for statistical data analysis. follicles in the paracortex (Fig. 2B). All follicles comprised an FDC network expressing MAdCAM-1, FDC-M2, or CD35 (Fig. Results 2B). MAdCAM-1+ MRCs lined the subcapsular sinus of the outer Rank-Tg mice display prominent postnatal LN growth margin of cortical follicles, with additional MRCs almost fully Mice overexpressing murine Rank under control of the S100A8 encircling the small paracortical follicles of the hyperplasic LNs promoter showed a massive hyperplasia of skin-draining LNs (Fig. 2B). To assess whether cLN hyperplasia led to changes in the (inguinal, axial, brachial, and superficial parotid), whereas internal proportions of FRCs, BECs, and LECs, we compared their num- LNs (mesenteric or para-aortic), spleen, and thymus showed no bers and distribution in cLNs of Tg versus WT animals. The cells were identified on the basis of podoplanin and CD31 expression such growth (Fig. 1A,1B). Cell counting revealed an 8–10-fold 2 increase in hematopoietic cell numbers of cLNs, whereas the after gating on CD45 cells (12). FRCs and BECs were present in counts for mesenteric LNs (mLNs), spleen, thymus, blood, and normal proportions, but the percentage of LECs showed a slight BM were unchanged or even slightly reduced (Fig. 1B, Table I). decrease (Fig. 2C). Absolute numbers demonstrated a robust in- To determine whether the SLO hyperplasia was acquired postna- crease in the abundance of all stromal subsets in the hyperplasic tally, we assessed cLN cell numbers 1 wk after birth and at dif- LNs. The subsets were normally distributed, with FRCs dispersed ferent time points thereafter. Growth started after week 2 and around cortical BECs, and LECs juxtaposed to B cell follicles reached a plateau at week 13 (Fig. 1C). To better determine its (Fig. 2D). Peripheral LN addressin-expressing high endothelial onset, we used in vivo ultrasound imaging, which measures LN venules were abundant and normally located in interfollicular regions (Fig. 2D). Thus, the cLN cell composition and its archi-

size in young mice more accurately than does cell counting or Downloaded from organ weighing (39). It ﬁxed the start of inguinal LN (iLN) growth tecture showed remarkably little alteration for such a signiﬁcant at P24 (Fig. 1D). Thus, Rank-Tg mice displayed a prominent growth, with the exception of an increase in the number and lo- postnatal growth of their cLNs. cation of B cell follicles and their associated stromal compartment. Conserved LN architecture with a notable increase in small B cell follicles The expanded immune compartment is not activated and

To investigate whether cLN hyperplasia was accompanied by responds to immunization http://www.jimmunol.org/ abnormal overrepresentation of a hematopoietic cell lineage, we To address the question of whether the hematopoietic cells of the determined the proportion of the CD11b+ granulo-myeloid lineage, cLNs were functional, we studied the cutaneous immune response. B220+ B cells, and CD3+ T cells by flow cytometry in cLNs, Skin- and blood-derived DCs, purified from brachial and axial mLNs, and spleen of Tg and control animals. There was a signif- LNs and identified on the basis of I-A/CD11c expression (41, 46), icant increase in CD11b+ cells in both LN types and the spleen in showed low CD86 levels but normally upregulated this activation mutant mice (Fig. 2A). An overrepresentation of these cells had marker in response to s.c. CpG injection (Fig. 3A). T cells of also been noted in the BM (44); therefore, it is most likely the cLNs, mLNs, or spleen were resting but responded to CpG by result of transgene expression by this lineage (44, 45) (data not CD62L downregulation and CD44 upregulation (Fig. 3B). Also, by guest on October 2, 2021 shown). More restrictively, there was an increase in the proportion B cells showed no sign of activation in nonimmunized Tg mice of B cells and a reduction in T cells in cLNs but not in mLNs or (Fig. 3C) and were activated in response to s.c. immunization by spleen. B cells do not express the transgene (45) (data not shown), OVA with LPS, CpG, or IFA (Fig. 3D). Of note, Ab-affinity suggesting that the change in B/T cell proportions was a reflection maturation to NP-KLH in LPS proceeded faster in Tg versus of the abnormal LN size. There was little alteration in B cell control mice (Fig. 3E). To test for a possible rupture of self- hematopoiesis, because the number of splenic transitional type 1 tolerance, we assessed the presence of serum autoantibodies and type 2 B cells remained relatively stable in comparison with against actin, rheumatoid factor, and DNA in both nonimmunized a strong reduction in mature B cells (Table II), suggesting a rein- and immunized Tg mice. However, no such Abs were detected forced recruitment of mature B cells to the cLNs. Therefore, we (data not shown). In addition, there was no significant difference visually inspected B cell organization in the cLNs by labeling in B cell numbers in the autoimmune-sensitive salivary glands sections for B220 and CD3. We noted normal B cell organization (WT: 30 6 12 3 103, Tg: 35 6 9 3 103; n = 8). In accord with

FIGURE 1. Postnatal cLN hyperplasia in Rank-Tg mice. A, Representative photographs of cLNs (left to right: inguinal, axial, brachial, and superﬁcial parotid LN) and an mLN from 12-wk-old WT and Rank-Tg mice. Two-fold magniﬁcation. B, Fold difference in cell numbers of cLNs, mLNs, spleen, thymus, blood, and BM in 12-wk-old Rank-Tg versus WT mice. C, cLN cell numbers from week 1 to 16 in Rank-Tg and WT mice. D, iLN surface area, measured by ultrasound, in Rank-Tg and WT mice from P4–P58. Insets show iLN ultrasound images of 12-wk-old WT and Tg mice. Data in B are from 11 mice, collected in three experiments; in C and D, data are mean 6 SD from six mice/genotype for each time point. **p , 0.01, ***p , 0.001. 1248 SECONDARY LYMPHOID ORGAN GROWTH BY RANKL

Table I. Absolute cell numbers in organs (106)

Animal cLNs mLNs Spleen Thymus Blood BM Rank-Tg (n = 11) 196 6 39 6.3 6 3.1 76 6 14 96 6 44 5.6 6 4.8 5.6 6 4.8 WT (n =8) 226 3 7.2 6 1.6 105 6 20 125 6 44 6.2 6 4.0 6.2 6 4.0 p , 0.001 NS p ,0.01 NS NS NS Cell numbers (mean 6 SD) of organ and blood cell suspensions from 12-wk-old Tg and WT animals were counted in a hemacytometer. these results, the proportion of Foxp3+ regulatory T cells was not BM of congenic CD45.1+ C57BL/6 mice; the reciprocal transfer markedly altered in cLNs, mLNs, or spleen (data not shown). was also done. cLN cell numbers were determined 3 mo later. BM Stromal cells express Aire, which is under RANK control in the transfers were complete, because all hematopoietic cells, includ- thymus (32–35), and regulate peripheral tolerance by expression ing the CD11b+ lineage, were of donor origin (Supplemental Fig. of tissue-restricted peripheral tissue Ags (PTAs) (5, 47–49). 1A,1B). WT → Tg chimeras developed LN hyperplasia to the Therefore, we evaluated Aire and PTA expression in Tg mouse same extent as did control Tg → Tg animals (Fig. 4A). Recipro- cLNs by qRT-PCR. No upregulation of Aire expression in CD452 cally, Tg BM could not transfer LN growth to WT mice. These stromal cells was seen (relative expression level, WT: 1, Tg: 0.642 6 ﬁndings demonstrated that Rank transgene expression by the 0.15; p = 0.092), and the stromal subset-restricted expression of granulo-myeloid cell lineage is not responsible for cLN growth.

Mlana and tyrosinase PTAs (48, 49) was also conserved (data not We determined whether the radioresistant stromal cells expressed Downloaded from shown). Therefore, postnatal LN growth has no discernable neg- the Rank transgene by performing RT-PCR on cLNs recovered ative inﬂuence on its immune cells, and peripheral tolerance is from WT → Tg chimeras. The transgene was no longer expressed, maintained. demonstrating that the cLN stroma is not transgenic for Rank (Supplemental Fig. 1C). We then considered the possibility that LN growth is associated with the transgenic HF cLN growth is conveyed by Rank transgene expression in HFs

To elucidate the mechanisms for organized cLN expansion, we (37). To address this issue, we ﬁrst transplanted Tg or WT skin http://www.jimmunol.org/ investigated the contribution of the Rank transgene in the CD11b+ from 3-wk-old donors onto the backs of age-matched nude mice. granulo-myeloid cell lineage by performing adoptive BM trans- Seven weeks later, cell numbers of brachial and axial LNs versus fers. Lethally irradiated 3-wk-old Tg mice were reconstituted with iLNs were determined. Because iLNs are not expected to drain by guest on October 2, 2021

FIGURE 2. The enlarged cLNs display a well-conserved cell composition and organ architecture. A, Cell lineage proportions in cLNs, mLNs, and spleen (S) of CD11b+ granulo-myeloid cells, B220+ B cells, and CD3+ T cells in Rank-Tg versus WT mice were identified by flow cytometry (n = 11/genotype). B, Representative images of T cell zone and B cell follicle organization (CD3 and B220 staining), MRCs (MAdCAM-1), and FDCs (MAdCAM-1, FDC-M2, CD35) in brachial LNs of Rank-Tg and WT mice. Scale bars, 100 mm; insets,50mm. C, Identification of the CD452 FRC, LEC, and BEC stromal cell subsets by flow cytometry, using podoplanin and CD31 cell surface markers. The percentage 6 SD is indicated next to the cell type. Graph (lower panel) depicts proportional changes. Table (lower panel) shows their absolute numbers in pooled inguinal, axial, and brachial LNs of Rank-Tg versus WT mice. Data are from 11–14 mice, accumulated in four experiments. D, Representative images showing BEC/FRC (CD31/podoplanin), LEC/B cell (Lyve-1/B220), and LEC/BEC (CD31/peripheral LN addressin) distribution on a brachial LN section. Scale bars, 100 mm; insets,50mm. *p , 0.05, **p , 0.01. The Journal of Immunology 1249

Table II. Absolute cell numbers of hematopoietic cell types (106)

CD11b+ Mature B T2 B T1/MZ B Animal Organ CD3+ T Cells Macrophages B220+ B Cells IgDModIgMMod IgDHiIgMHi IgDLoIgMHi Rank-Tg (n = 11) cLNsa 53.9 6 17.7 2.7 6 1.2 65.4 6 30 WT (n = 8) 8.5 6 3.1 0.2 6 0.06 5.6 6 2.3 p , 0.001 p , 0.001 p , 0.001 Rank-Tg (n = 11) mLNs 3.3 6 1.4 0.08 6 0.07 2.3 6 1.3 WT (n = 8) 4.3 6 1.1 0.07 6 0.06 2.4 6 0.5 NS NS NS Rank-Tg (n = 11) Spleen 16.4 6 4.7 4.4 6 2 36.1 6 9.5 28 6 12 2.6 6 1.6 8.8 6 4.5 WT (n = 8) 25.4 6 6.1 3.3 6 0.6 57 6 13 39 6 9 3.6 6 0.6 7.4 6 3.4 p , 0.001 NS p , 0.002 p , 0.03 p , 0.05 NS Rank-Tg (n = 11) Blood (ml) 2.2 6 1.3 1.4 6 1.0 2.9 6 2.2 WT (n = 8) 1.7 6 0.7 1.0 6 0.4 2.7 6 1.5 NS NS NS Cell numbers (mean 6 SD) of organs and blood cell suspensions were counted in a hemacytometer and analyzed for T cells, macrophages, B cells, and B cell subsets by flow cytometry. The total number was determined using the percentage composition of the different cell types. acLNs included inguinal, axial, brachial, and superficial parotid; for B cells and macrophages, cell numbers were determined excluding brachial LNs. T1, transitional type 1, T2, transitional type 2, MZ, marginal zone. Downloaded from from the graft, their cell counts served as internal control for large arrows). This underscores the importance of MRCs in LN natural LN size and experimental variations. We found a signifi- homeostasis and suggests a role for RANKL in MRC mainte- cant increase for Tg skin recipients compared with WT skin, in- nance. RANKL neutralization did not lead to a reduction in dicating that LN growth is associated with Rank transgene splenocyte numbers (Supplemental Fig. 2C) nor were B or T cells expression in HFs (Fig. 4B). We verified this conclusion by re- activated, as assessed by plasmocyte and memory T cell formation ducing transgene expression in HFs. This was done by crossing (Supplemental Fig. 2D,2E). The anti-RANKL treatment provoked http://www.jimmunol.org/ the transgene onto Msx-2lacZ/lacZ mice, which have a shortened a diminution in body weight (WT: 17.7 6 0.5 g, Tg: 18.7 6 1.1 g, anagen phase (50) and, therefore, are expected to express lower treated Tg: 12.1 6 1.6 g; n = 12), most probably attributable to levels of the Rank transgene (Supplemental Fig. 1D). This mutant perturbation of bone homeostasis (25, 26). These data showed that gene combination resulted in strongly reduced cLN growth (Fig. RANKL is responsible for LN growth and formation of small 4C). To exclude the possibility that Msx-2 affected the organ size B cell follicles in the Rank-Tg mouse. through local expression, we assessed b-galactosidase activity in RANKL activates chemokine synthesis and proliferation of SLO brachial LNs of Rank Tg;Msx-2lacZ/+ animals. lacZ gene activity stroma

was not detected in the LNs, but it was found at the base of the by guest on October 2, 2021 HFs (Fig. 4D) (50). Taken together, these findings supported the To further address the mechanism of LN growth, we first con- conclusion that Rank transgene expression in HFs is necessary and sidered adult LTi-like cells as a possible RANKL target. They have sufficient for skin-draining LN hyperplasia. been shown to express RANK, and their role in stroma activation could depend on RANK–RANKL interactions (27, 53). Moreover, RANKL induces cLN growth their implication in SLO expansion was observed in the case of To identify the signal that is conveyed by the anagen HF and that is enhanced survival by ectopic IL-7 expression (54). We compared responsible for cLN growth, we considered RANKL as a probable adult LTi-like lin2Rorgt+CD127+a4b7+ cells of WT and Rank-Tg candidate, because the anagen HF is an important source of soluble mice in terms of phenotype, numbers, and frequency. Moreover, RANKL (37); soluble RANKL is a small (.10 kDa) globular because RANK stimulates LTa1b2 expression by LTi cells (28), protein, which can be expected to have direct access to LN stroma we measured expression of LTa1b2. The experiments were per- through its conduit system (3, 51, 52); and RANKL is known to formed at P24 and P42, shortly after skin RANKL peaks and play a critical role in SLO formation (25–27). We measured sol- initiation of LN growth. No significant difference in cell numbers, uble RANKL and OPG release from Tg and WT skin at different phenotype, or LTa1b2 expression was noted (Fig. 6A, Supple- time points during HF morphogenesis and cycling. We confirmed mental Fig. 2F). We also measured LTa1b2 expression by B cells that RANKL was highest at anagen, when OPG levels were low but did not find a significant difference (Fig. 6A). We then studied (37). Moreover, higher levels of RANKL were released from Tg FRC and endothelial cells, in view of their role in coordinating skin at P14 and during anagen (Supplemental Fig. 2A,2B). We lymphoid organ formation (2, 4–6). First, we determined Rank then directly tested the role of RANKL by s.c. administration of expression by RT-PCR on FACS-sorted FRCs, LECs, and BECs. a RANKL-blocking mAb. Tg mice received the mAb from P10 to Rank was transcriptionally active in all stromal subsets (Fig. 6B), wk 6, whereas littermates were administered control mAb. cLNs suggesting that skin-derived RANKL could directly stimulate and spleen were weighed and cells counted, and B/T cell pro- cLN stroma. Because chemokines and cell-adhesion molecules are portion was assessed. The RANKL-neutralizing mAb restored expressed by stroma and play an important role in SLO growth cLN weight and cell numbers (Fig. 5A). The proportion of B cells (9, 55), we tested whether these type of molecules were upregu- was reversed (Fig. 5B). The number of B cell follicles was related. We performed qRT-PCR on sorted stromal subsets harvested covered, and their size was restored (Fig. 5C). We then determined from cLNs of 6-wk-old WT and Tg animals. Among the different the presence of MRCs by labeling sections for MAdCAM-1 and genes tested (Supplemental Table I), we found in Tg mice that discovered a distinct reduction (thin arrows, mouse #1) or even FRCs expressed more CXCL13 transcripts; LECs expressed more a complete absence of these B cell-associated stromal cells CCL19, MAdCAM-1, and VCAM-1 transcripts; and BECs ex- (mouse #2). In contrast, FDCs remained intact, as shown by la- pressed more CCL19 transcripts (Fig. 6C). This suggested that beling for MAdCAM-1 or FDC-M2 within the follicles (Fig. 5D, cLN growth is promoted by RANK-stimulated expression of 1250 SECONDARY LYMPHOID ORGAN GROWTH BY RANKL Downloaded from

FIGURE 4. Tg RANK overexpression in HFs induces cLN hyperplasia. http://www.jimmunol.org/ A, Analysis of cell numbers in cLNs (brachial, inguinal, and axial) after adoptive BM transfer. Data are from three mice/group compiled from seven different experiments. B, Relative cLN cell number increase in brachial and axial LNs, normalized to iLNs in nude mice after skin grafting. Data are from three mice/group compiled from three different experiments. C, cLN cell numbers in WT, Msx-2LacZ/LacZ, Rank-Tg, and Rank-Tg;Msx-2LacZ/LacZ mice. Data are from eight mice/group from four experiments. D, Representative b-galactosidase staining in a brachial LN LacZ/+ (left panel) and skin (right panel)ofRank-Tg;Msx-2 mice. Arrow- by guest on October 2, 2021 heads point to b-galactosidase enzymatic reactivity of HF matrix cells. Dashed line indicates the dermo–epidermal junction. Scale bar, 100 mm. Data are mean 6 SD. *p , 0.05, ***p , 0.001.

cell proliferation (35), we next asked whether stromal cells pro- FIGURE 3. cLN immune cells are resting but respond rapidly to immu- liferated in response to RANKL. To this end, we measured cell nization. A, CD86 expression levels of skin- and blood-derived DCs, purified division by BrdU incorporation in 6-wk-old WT mice, as well as from brachial and axial LNs in nonimmunized WT and Rank-Tg mice, as mock- and anti-RANKL–treated Tg mice. Mice were given BrdU well as from Rank-Tg mice 4 d after a s.c. immunization boost with CpG. for 6 d, and DNA incorporation of the nucleotide analog was Graph (left panel) shows the mean 6 SD of three animals/genotype per- measured by flow cytometry. The WT CD452 stroma had a rea- formed in duplicates. The flow cytometry dot blot (right panel) depicts the sonable proliferation rate, but it was much faster in Tg animals. Hi + + Hi gate setting for I-A CD11c skin-derived DCs and for I-A CD11c Strikingly, when RANKL was neutralized, the proportion of blood-derived DCs. B, Percentage of CD44- and CD62L-expressing Th cells BrdU+ stroma decreased to WT levels (Fig. 6D). In contrast, the in cLNs, mLNs, or spleen in WT, Rank-Tg, and Rank-Tg mice 4 d after a s.c. immunization boost with CpG (left panel). Data are mean 6 SD of six to BrdU incorporation rate for hematopoietic cells was similar be- eight animals/genotype. The flow cytometry dot plots (right panel) depict tween WT and Tg animals, and there was no change after ad- a representative analysis of CD62L and CD44 expression of nonimmunized ministration of the blocking mAb. We next analyzed FRCs, LECs, and CpG-immunized WT and Tg Th cells from brachial LNs. C,CD86and and BECs separately. All subsets proliferated faster in Tg mice, CD69 expression levels of B220+ B cells in cLNs, mLNs, and spleen from and their greater BrdU incorporation was abrogated upon RANKL WT and Rank-Tg mice. Data are mean 6 SEM of five animals/group. D, neutralization (Fig. 6E). Pulse-chase experiments were performed Serum anti-OVA IgG levels in WT and Rank-Tg mice, measured after a s.c. to address stromal cell viability. Loss of the BrdU label was not immunization boost of OVAwith LPS, CpG, or IFA. Each symbol represents delayed in Tg mice, indicating that RANKL promoted stromal cell one animal, and the horizontal lines show the mean values. E, High-affinity division without altering cell viability (Supplemental Fig. 2G). anti-NP IgG levels after s.c. immunization of NP conjugated to KLH with 30 Thus, RANKL stimulates cLN growth by inducing the prolifera- LPS. Serum anti-NP IgG was measured by ELISA for NP -BSA or NP - 3 30 tion of its vascular and reticular fibroblastic stromal cells. BSA, and the relative affinity was determined as the titer NP3/titer NP30. Data are mean 6 SEM of seven mice/group. **p , 0.01, ***p , 0.001. Discussion chemokines and adhesion molecules. In light of the almost un- In this study, we showed that RANK stimulation resulted in the changed stromal cell proportion of the enlarged cLNs, together proliferation of LN stromal subsets and their expression of che- with the finding that RANKL induced medullary thymic epithelial mokines and adhesion molecules, culminating in LN growth. LN The Journal of Immunology 1251

FIGURE 5. RANKL induces cLN growth. A, Weight (left panel) and cell numbers (right panel) of cLNs, normalized to body weight, of WT, control Ig-administered Rank-Tg mice, and Rank-Tg mice, treated with anti-RANKL blocking mAb from P10 to 6 wk of age. B, Percentage of T and B cells in cLNs of WT, control Ig-administered Rank-Tg mice, and Rank-Tg mice, treated with anti-RANKL blocking mAb. C, Left panel, Representative visu- alization of B cell follicles in WT mice, control Ig- administered Rank-Tg mice, and Rank-Tg mice, treated with anti-RANKL blocking mAb by im- munoﬂuorescence on axial LN sections. Scale bars, 200 mm. B cell follicles visualized on B220-labeled axial LN cross-sections were counted (middle panel) and their area was quantiﬁed (right panel). Data are mean 6 SEM of nine mice/group acquired in three separate experiments. D, MAdCAM-1+

MRCs in LNs of mock-treated and anti-RANKL- Downloaded from treated Rank-Tg mice (mouse #1 and #2). Arrows indicate MRCs at the outer margin of the B220+ B cell follicles. FDC-M2+ FDC networks on serial sections are indicated by large arrows. Dashed lines mark the limit of the LNs. Scale bars, 100 mm. **p , 0.01, ***p , 0.001. http://www.jimmunol.org/

organization was preserved, with a notable increase in B cell that MRCs are implicated in de novo B cell follicle formation. In follicles. RANK stimulation did not provoke an immune activation; support of this is the loss of MRCs, as well as B cells, after RANKL no autoreactivity could be detected, and immune cells responded neutralization. rapidly and strongly to immunization by foreign Ags. The greatly expanded SLO size could have been the conse- Although recognized as an important regulator of immunity, the quence of a dysfunctional hematopoietic cell compartment. However, by guest on October 2, 2021 lack of almost all SLOs in Rank or Rankl KO mice has hampered T cells, B cells, and DCs showed no signs of activation, and no elucidation of RANK function in the development and homeostasis autoantigen reactivity could be detected. Moreover, T cells did not of the immune system. Therefore, we studied a Rank-Tg mouse, spontaneously proliferate in culture, as described in a different which displayed greatly oversized skin-draining LNs, whereas other model of SLO hyperplasia (60), and transfer of cLN CD4+ T cells SLOs, spleen, thymus, or BM were normal. LN growth occurred into Rag-2 KO mice did not result in abnormal expansion (data not postnatally and, at its plateau, the lymphocyte number surpassed shown). The cLN immune cells of the Rank-Tg animals were not that of the spleen. We could not detect other major abnormalities of anergic, because they responded to immunization. The immune the immune system, aside from a general overrepresentation of response elicited from the Tg mice was even moderately superior Rank-Tg CD11b+ cells, presumably attributable to the cell survival to that of WT mice, and a more rapid Ab-affinity maturation was action of RANK (56–58). A condition that resembles this pheno- seen. The increased efficiency of the humoral response may reflect type is found in a IL-7–Tg mouse (54). In this mutant, SLO growth increased numbers of B and T cells, as well as more B cell fol- and ectopic SLO formation were linked to increased LTi cell num- licles comprising MRCs and FDCs. Despite the relationship be- bers. Dissimilar to the IL-7–Tg mouse model, the SLO expansion tween RANKL and thymic epithelial cells (32–35), and although described in this article appeared much more prominent, and no LN Aire appears to contribute to peripheral T cell tolerance (49, 61), ectopic SLOs could be found. Despite massive organ growth, we could not detect such an association between RANKL and Aire hematopoietic and stromal cell compositions, as well as the overall in cLN stroma of the Tg animals. architecture, were remarkably well conserved. The only notable By performing BM transfer, skin transplantation, and genetic alterations were increases in the number of small B cell follicles and rescue experiments, we discovered that the SLO growth signal the proportion of B cells. Support for a role for RANK signaling in originated in the HF. This finding provides an explanation for why regulating peripheral immune cell numbers stems from a study of only skin-draining LNs were affected and is in accord with initi- Opg2/2 mice. In these animals, LN B and T cell numbers were ation of LN growth at the start of the HF anagen phase after P24. likewise augmented, and the B cell compartment was boosted, The growth plateau is likely due to loss of transgene expression in particularly in the spleen (59). This phenotype was attributed to an the HF as the mouse develops alopecia (37). It is interesting to note accelerated B cell development. We did not find a prominent in- that the postnatal period during which skin-draining LN expansion crease in splenic transitional type 1 or type 2 B cells, discounting the surpasses body growth occurs during HF morphogenesis and the possibility that the additional cLN B cells are the result of acceler- first anagen phase (39). This is suggestive of a naturally operating ated B cell hematopoiesis. Instead, the higher B cell proportion is link between HFs and cLNs, an association that appears to be likely due to reinforced recruitment or maintenance of mature amplified in the Tg mouse. We considered the possibility that B cells. Formation of the paracortical follicles was accompanied by DCs mediate the HF–LN link; however, in analogy to derma- colonization of MRCs in their outer margin, evoking the proposal topathic lymphadenopathy (62), more numerous, long-lived, or 1252 SECONDARY LYMPHOID ORGAN GROWTH BY RANKL

be expected to have unobstructed access to all stromal cells through their conduits (3). We found that RANKL neutralization by a blocking mAb restored normal LN size, B/T cell ratio, and follicle area and numbers. We cannot formally exclude the possibility that blocking RANKL from other sources contributed to normalization; however, skin RANKL was deﬁnitely affected, because abnormal HF cycling and epidermal dysplasia were cor- rected (37). Loss of B cells and reduction of FRC proliferation beyond WT levels in the treated mice may be due to blocking RANKL produced by MRCs (7). Indeed, because RANKL neutralization led to the disappearance of MRCs from the preserved B cell follicles, a role for endogenous RANKL in the maintenance of MRCs can be evoked. Among other potential sources of SLO RANKL, we can exclude T cells, which synthesize RANKL only when activated (57). Although the mAb was administrated s.c., bone homeostasis was affected, because the treated mice displayed growth retardation. However, a disturbed hematopoietic cell output by reduction of BM niches (63) was not seen, because spleen cell numbers were not diminished. In a different model of skin RANKL overexpression by Rankl transgenesis under control Downloaded from of the keratin 14 promoter, cLN hyperplasia was not reported (64). Although this difference may suggest that factors other than RANKL are implicated in cLN growth, the cardinal difference between the two mouse models is that RANKL is not soluble in keratin 14 promoter Rankl Tg mice and, therefore, cannot freely reach the draining LNs. As proposed by the investigators, it is http://www.jimmunol.org/ FIGURE 6. RANKL activates stromal cell proliferation and chemokine/ likely that the Tg RANKL principally affects Langerhans cells. adhesion molecule expression. A, Left panel, Adult LTi-like cells (lin2 To identify the cellular RANKL target, we considered adult LTi- RORgt+a4b7+CD127+) were identiﬁed in LNs of WT and Rank-Tg mice at like cells and stromal cells as candidates. However, the proportion

P24 and P42, and absolute numbers were determined. Middle panel,LTa1b2 of adult LTi-like cells was not altered, nor was the phenotype and mean fluorescence intensity of adult LTi-like cells. Right panel, Relative expression of LTa1b2 by adult LTi-like cells or B cells different. expression levels of LTa1b2 mRNA in FACS-sorted adult LTi-like cells and We also could not detect an increase in IL-7 synthesis by FRCs, + CD19 B cells at P42 (n = 6 animals). B, RT-PCR analysis of Rank and an important SLO source of IL-7 (12) (data not shown). We could GAPDH mRNA expression by stromal subsets from cLNs of WT and Rank- also rule out Rank transgene expression by the radioresistant by guest on October 2, 2021 Tg mice, sorted by flow cytometry on the basis of podoplanin/CD31 ex- stromal cells, because its mRNA was no longer detectable after pression. Skin cDNA served as positive control, and water served as negative WT → Tg BM transfer. However, all stromal subsets expressed control. Image is representative of four separate experiments with 15 pooled WT mice and 5 pooled Tg mice. C, mRNA expression of chemokines and endogenous RANK. It is known that endothelial cells (65–67) and adhesion molecules in flow cytometry-sorted stromal subsets from WT and mesenchymal cells (56, 68) express RANK, but whether their Rank-Tg mice. Measurements were done by qRT-PCR and are depicted as SLO counterpart likewise carries RANK has not been studied. The the fold increase with respect to WT. Data are the mean 6 SEM of four in- cLN stroma upregulated chemokines and cell-adhesion molecules; dependent experiments performed in duplicate with 15 pooled WT mice and however, more strikingly, it showed a dramatic hyperproliferation 5 pooled Rank-Tg mice. D, BrdU incorporation measured by flow cytometry in the Tg mice, which was fully abolished by RANKL neutrali- 2 + of CD45 cLN stromal and CD45 hematopoietic cells from WT, control Ig- zation. Because of podoplanin coexpression it was not possible administered Rank-Tg mice, and Rank-Tg mice treated with anti-RANKL to differentiate between TRCs and MRCs within the FRC pop- blocking mAb. E, BrdU incorporation by cLN stromal subsets from WT, ulation. Other attempts to identify MRCs within the FRC pop- control Ig-administered Rank-Tg mice, and Rank-Tg mice treated with anti- ulation were not successful because of technical challenges, likely RANKL blocking mAb. Data in D and E are mean 6 SEM of six to nine mice per group, obtained by flow cytometry using podoplanin/CD31 markers to caused by the incompatibility of enzymatic liberation of cells identify the subsets. **p , 0.01, ***p , 0.001. with preservation of cell-adhesion molecules, such as MAdCAM-1. It is also possible that MRCs are concealed within the CD45+ cluster, given their close association with B cells (7). However, if mature DCs would principally lead to an expanded T cell zone. MRCs were the only RANK-expressing cell type, one could ex- This was not seen, and the proportion of DCs and their maturation pect a specific enrichment of MRCs versus TRCs. The unchanged status were unchanged. Because shortening of anagen by loss of qRT-PCR measures of Rankl mRNA, expressed by MRCs but not Msx-2 abrogated LN growth, we deduced that the signal arises TRCs (7) (data not shown), do not support a scenario of MRC- during the anagen phase of the hair cycle. High levels of soluble restricted proliferation. Whether FDCs also express RANK is RANKL released from skin prompted us to propose the concept uncertain, owing to the known difficulties with obtaining a suffi- that HF RANKL conveys cLN growth. In fact, the level of HF ciently homogeneous FDC population. RANK-mediated FDC RANKL is higher in the mutant animal, and the skin undergoes proliferation would support the increase in B cell follicles. Indeed, additional anagen phases, leading to a continuous RANKL release. the Rank gene was expressed in a transcriptome study of FDCs The underlying reason for the increase in HF RANKL is unclear. (69) (C. Berek, personal communication). However, in keeping One possibility is the existence of a self-amplifying RANK– with the idea that MRCs may be FDC precursors (7), it remains RANKL loop, similar to what was described for thymic epithelial a possibility that RANK stimulation of MRC translates into de cells (36). Another argument in favor of RANKL as mediator of novo B cell follicle formation comprising FDCs. We observed that SLO growth is that RANKL is a small globular protein, which can the proportion of LECs is diminished in Tg LNs, although pro- The Journal of Immunology 1253 liferation and turnover rate are similar to those of BECs/FRCs. 6. Luther, S. A., T. K. Vogt, and S. Siegert. 2011. Guiding blind T cells and den- . dritic cells: A closer look at fibroblastic reticular cells found within lymph node Occasionally, older mice ( 20 wk) present with lymphedema in T zones. Immunol. Lett. 138: 9–11. cLNs. This suggests an impaired function in the LEC compart- 7. Katakai, T., H. Suto, M. Sugai, H. Gonda, A. Togawa, S. Suematsu, Y. Ebisuno, ment, so that reduction of egress rates may contribute to LN hy- K. Katagiri, T. Kinashi, and A. Shimizu. 2008. Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs. J. Immunol. 181: 6189–6200. perplasia (11). The reasons for the loss of LECs are unclear and 8. Palframan, R. T., S. Jung, G. Cheng, W. Weninger, Y. Luo, M. Dorf, necessitate further investigations. D. R. Littman, B. J. Rollins, H. Zweerink, A. Rot, and U. H. von Andrian. 2001. It was found that RANK ligation on vascular cells stimulates Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues. J. Exp. angiogenesis and ICAM-1 and VCAM-1 expression (66, 67, 70). In Med. 194: 1361–1373. an artificial SLO-like cell system, high fluid pressure activates 9. Browning, J. L., N. Allaire, A. Ngam-Ek, E. Notidis, J. Hunt, S. Perrin, and stromal cell chemokine synthesis (71). This invites speculation on R. A. Fava. 2005. Lymphotoxin-beta receptor signaling is required for the ho- meostatic control of HEV differentiation and function. Immunity 23: 539–550. the existence of an interlinked direct RANK action on endothelial 10. Pham, T. H., P. Baluk, Y. Xu, I. Grigorova, A. J. Bankovich, R. Pappu, cells and reticular fibroblasts. Importantly, LTbR and TNFR sig- S. R. Coughlin, D. M. McDonald, S. R. Schwab, and J. G. Cyster. 2010. 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