A Stroma-Derived Defect in NF-κB2−/− Mice Causes Impaired Lymph Node Development and Lymphocyte Recruitment

Damian Carragher, Ramneek Johal, Adele Button, Andrea White, Aristides Eliopoulos, Eric Jenkinson, Graham Anderson and Jorge Caamaño
J Immunol August 15, 2004, 173 (4) 2271-2279; DOI: https://doi.org/10.4049/jimmunol.173.4.2271

Abstract

The NF-κB family of transcription factors is vital to all aspects of immune function and regulation in both the hemopoietic and stromal compartments of immune environments. Recent studies of mouse models deficient for specific members of the NF-κB family have revealed critical roles for these proteins in the process of secondary lymphoid tissue organogenesis. In this study, we investigate the role of NF-κB family member NF-κB2 in lymph node development and lymphocyte recruitment. Inguinal lymph nodes in nfκb2−/− mice are reduced in size and cellularity, most notably in the B cell compartment. Using in vitro and in vivo lymph node grafting assays, we show that the defect resides in the stromal compartment. Further examination of the nfκb2−/− inguinal lymph nodes revealed that expression of peripheral node addressin components CD34 and glycosylation-dependent cell adhesion molecule-1 along with the high endothelial venule-restricted sulfotransferase HEC-GlcNAc6ST was markedly reduced. Furthermore, expression of the lymphocyte homing chemokines CCL19, CCL21, and CXCL13 was down-regulated. These data highlight the role of NF-κB2 in inguinal lymph node organogenesis and recruitment of lymphocytes to these organs due to its role in up-regulation of essential cell adhesion molecules and chemokines, while suggesting a potential role for NF-κB2 in organization of lymph node endothelium.

Lymph nodes (LN)3 and Peyer’s patches (PP) are highly organized secondary lymphoid organs where the essential interactions between lymphocytes and dendritic cells take place during the initial steps of adaptive immune responses.

Among the early events that give rise to LNs during embryonic development is the transendothelial migration of CD45+CD4+CD3−IL-7Rα+α4β7+ cells (inducer cells) to colonize LN anlagen (for review on inducer cells, see Ref.1). Inducer cells express high levels of the membrane-bound ligand lymphotoxin α1β2 (LTα1β2), which upon binding to LTβR on mesenchymal cells (organizer cells) stimulates expression of VCAM-1, ICAM-1, and the homing chemokines CXCL13/B lymphocyte chemoattractant, CCL19/EBV-induced molecule 1 ligand chemokine, and CCL21/secondary lymphoid tissue chemokine (2, 3). These chemokines induce a feedback loop that attracts large numbers of further inducer cells expressing the CXCL13 receptor, CXCR5, as well as the receptor for CCL19 and CCL21, CCR7 (3, 4). Clustering of these inducer cells with stromal cells of the LN anlagen is a vital part of further productive LN development (5, 6) (for review, see Ref.7).

A few days after birth, a switch in the expression of adhesion molecules in high endothelial venules (HEVs) occurs. Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) levels are reduced, and peripheral node addressin (PNAd) expression is observed, facilitating the homing of L-selectin+ lymphocytes to LNs (8). The chemokines CCL21, CCL19, CXCL13, and CXCL12/stromal cell-derived factor-1 together with PNAd participate in transendothelial migration of lymphocytes into LNs and PPs (9, 10). Lymphocyte recruitment creates a positive feedback loop between B cells and stromal cells mediated by CXCL13/CXCR5 signaling in the former and LTα1β2/LTβR in the latter (11).

Cross talk interactions between hemopoietic and stromal cells during LN development are mediated by LTα1β2/LTβR, TNF-related activation-induced cytokine (TRANCE)/TRANCE-R, TNF-α/TNF-RI, and CXCL13/CXCR5. Gene knockout experiments have revealed that ltα−/− and ltβr−/− mice lack all LNs and PPs, while ltβ−/− mice have only mucosal LNs (12, 13). Likewise, trance−/− mice present with rudimentary LNs containing a reduced number of inducer cells (5). Signaling through LTα1β2/LTβR and, to a lesser degree, LTα3/TNFR-I cooperates in the development of LNs and PPs (13, 14, 15).

Importantly, LTβR, TRANCE-R, and TNF-RI signaling pathways converge in activation of the NF-κB family of transcription factors. The Rel/NF-κB proteins regulate transcription of a large number of cellular genes involved in innate and adaptive immune and inflammatory responses (16, 17, 18). Mammalian cells express five NF-κB proteins: NF-κB1 (the DNA-binding subunit p50 and its precursor p105), NF-κB2 (the DNA-binding subunit p52 and its precursor p100), RelA (p65), RelB, and c-Rel. Nonstimulated cells contain cytoplasmic homo- and heterodimers of these factors bound to a family of IκBs. Activation of NF-κB occurs via at least two distinct pathways (19). Signaling through the TNF-RI and other proinflammatory cytokine receptors activates the classical NF-κB pathway through the IκB kinase (IKK) signalosome complex, containing the IκB kinases α and β (IKKα, IKKβ), and a regulatory subunit, IKKγ/NF-κB essential modulator, induces phosphorylation and subsequent degradation of IκBα facilitating nuclear translocation of p50/RelA dimers. In contrast, LTβR signals through both the classical and the alternative NF-κB pathway that through the NF-κB-inducing kinase (NIK) and an IKKα complex induces the processing of NF-κB2 p100 to p52 and the nuclear translocation of p52/RelB (20, 21, 22, 23, 24, 25, 26).

The NF-κB proteins have specific functions during secondary lymphoid tissue organogenesis (27). We and others have shown that nfκb2−/− mice present with marked defects in the architecture of the spleen, and lack follicular dendritic cells due to defects in stromal cells (28, 29, 30). Interestingly, relB−/− mice have similar defects due to impairment in stromal cells (31, 32). RelA has an important function in stromal cells during LN development, as shown by the absence of LNs in relA−/−/tnfr1−/− mice (33). Similarly, wnt-IκBα dominant-negative mice lack LNs, due to inhibition of Rel-A-containing complexes (34)

Abnormal splenic architecture, follicular dendritic cell maturation, and PP development in NIK (nik−/− mice and aly/aly mice) or IKKαaa kinase mutant mice demonstrated the role of these two kinases during the organization of secondary lymphoid tissues (35, 36, 37).

In this study, we analyze the role of NF-κB2 during LN development and lymphocyte recruitment to LNs. We show that nfκb2−/− mice have a defect in the development of inguinal LNs (ILNs) with a remarkable decrease of lymphocyte numbers. We have developed a series of in vivo and ex vivo assays to investigate whether NF-κB2 is required cell autonomously in bone marrow-derived or stromal cells during LN organization. We find that nfκb2−/− lymphocytes are able to populate wild-type ILNs, whereas nfκb2+/+ lymphocytes are dramatically impaired in their migration into nfκb2−/− ILNs due to an intrinsic defect in the stromal cells of these organs. Our results demonstrate that NF-κB2 has an important function in stromal cells in regulating the expression of the cell adhesion molecules and chemokines required for proper LN organogenesis and to drive lymphocyte transendothelial migration into these organs.

Materials and Methods

Mice

The generation of nfκb2−/− mice has been previously described (28). Mice were backcrossed >10 generations in C57BL/6J background. CD45.1 congenic mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All animals were bred and maintained at the Biomedical Service Unit of the University of Birmingham according to Home Office regulations.

LN dissection and immunostaining

LNs were dissected from the different mouse strains, embedded in OCT compound, snap frozen in liquid N2, and stored at −70°C until further use. Sections (5 μm) were cut using a cryostat and serially mounted onto multispot slides. Tissue sections were air dried, acetone fixed, and stored at −20°C. Slides were washed in Tris buffer, pH 7.6, and primary Abs were added and incubated at room temperature for 45 min. Abs used in this study were: anti-CD3, anti-IgD, anti-CD45.2, anti-MAdCAM-1, MECA-79 (BD Pharmingen, San Diego, CA), anti-CXCL13 (R&D Systems, Minneapolis, MN), and biotinylated-Lycopersicon Esculentum (tomato) lectin, which binds to N-acetylglucosamine groups. Slides were washed, and secondary Abs were added for 45 min. After further washes, the streptavidin-biotin complex was added for 30 min. Color was developed with diaminobenzidine peroxidase. Alkaline phosphatase was developed using Fast blue and Naphthol AS-MX phosphate.

Neonatal LN colonization assays

CD4+ cells were isolated from postnatal day 3 (P3) thymi by magnetic bead depletion of CD8+ cells, followed by staining for CD4 PE (BD Pharmingen), and positively sorted for PE+ cells using a magnetism-assisted cell-sorting column method (MiniMACS). The resulting population was analyzed for CD4 expression via FACS (BD Biosciences, San Jose, CA). Nfκb2−/− CD4+ cells were labeled with CFSE, while the nfκb2+/+ CD4+ cells were stained with PKH 2.6 (Sigma-Aldrich, St. Louis, MO). ILNs were excised from P2 nfκb2+/+ and nfκb2−/− mice and cultured in the presence of 1 × 105 labeled cells in a hanging drop system in Terasaki plates for 24 h before analysis by FACS or immunofluorescence on frozen sections.

Semiquantitative (Sq) RT-PCR

LNs were excised and disaggregated with trypsin/EDTA. The resulting cell suspension was depleted of CD45+ cells via magnetic depletion beads (Dynal Biotech, Great Neck, NY). Cells were snap frozen, RNA extraction was performed using TRIzol (Sigma-Aldrich), and cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Sq RT-PCR was undertaken on neonatal LN stroma or ILNs using primers that have been previously described (31, 38). PCR products were separated by electrophoresis in 1.8% agarose gels and visualized on a Syngene gene viewer.

Cell counts, FACS, and statistical analyses

Superficial inguinal, axillary, and mesenteric LNs were isolated, and a single cell suspension was prepared by teasing the organs with a needle and forceps. Cells were counted in the presence of trypan blue and labeled with mAbs anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-CD19, anti-CD45.1, anti-CD45.2, anti-IL-7Rα, and anti-annexin V (BD Pharmingen). Ten thousand events were collected and analyzed on a FACSCalibur (BD Biosciences). Cells were isolated from one ILN per mouse, and six mice per genotype were used in each assay. Statistical analyses were performed using the Mann-Whitney two-tailed test.

LN grafting

ILNs were excised from P2 nfκb2+/+ and nfκb2−/− mice and grafted by surgical procedure under the kidney capsule of 6- to 8-wk-old Ly-5.1 or Ly-5.2 C57BL6J nfκb2+/+ or Ly-5.2 C57BL6J nfκb2−/− mice. Grafts and host ILNs were extracted after 2 wk and analyzed by FACS analysis or immunostaining.

Results

Impaired LN development in nfκb2−/− mice

During the course of previous studies on nfκb2−/− mice, we observed that peripheral LNs, in particular inguinal and popliteal LNs of adult mice, were dramatically reduced in size compared with LNs of nfκb2+/+ littermates (Fig. 1⇓A). This fact prompted us to pursue a thorough analysis of these organs to understand the cause of this impairment. Further observation demonstrated that adult nfκb2−/− mice lack popliteal LNs. In addition, 40% of the adult nfκb2−/− mice analyzed (n = 20) have very rudimentary ILNs that contain mainly stromal cells and very few lymphocytes, which makes their study rather difficult. However, the remaining 60% of adult nfκb2−/− ILNs, although reduced in size, were large enough to allow further studies. Absolute cell numbers, but not percentages, of CD4+ and CD8+ T cells, were found to be reduced in the ILNs of nfκb2−/− mice compared with nfκb2+/+ littermates (Fig. 1⇓, B–D): the mean of CD4+ T cells is 6-fold lower in nfκb2−/− ILNs (nfκb2+/+, 8.49 × 105 cells SEM 1.39 × 105; nfκb2−/−, 1.38 × 105 cells SEM 2.53 × 104 (p < 0.0043)). Moreover, the number of B220+ B cells was found to be reduced 12-fold compared with nfκb2+/+ ILNs (nfκb2+/+, 2.78 × 105 cells SEM 5.7 × 104; nfκb2−/−, 2.24 × 104 cells SEM 3.8 × 103 (p < 0.0043)), as well as a reduction in the percentage of B cells also being noted (Fig. 1⇓, E and F). Annexin V staining and FACS analysis of cells recovered from ILN nfκb2−/− mice showed an increase in the frequency of cells undergoing apoptosis, compared with nfκb2+/+ mice (nfκb2+/+, 45.5% SEM 2.3; nfκb2−/−, 81.5% SEM, 5.5, n = 6, p < 0.0022) (data not shown). Immunohistochemical analysis of T and B cell distribution in the ILNs from adult nfκb2+/+ mice (Fig. 1⇓, G and I) and nfκb2−/− (Fig. 1⇓, H and J) mice shows that the latter form only small IgD+ B cell areas in the cortex of the LNs (Fig. 1⇓, G and H). Many nfκb2−/− B cells were scattered among CD3+ T cells in the paracortex of the LNs (arrowheads, Fig. 1⇓H). Large numbers of B cells were seen surrounding large HEVs in nfκb2+/+ ILNs, but only few B cells were observed around small-sized HEVs in nfκb2−/− ILNs (Fig. 1⇓, G and H). Furthermore, higher magnification of tissue sections shows that in contrast to the well-organized B cell areas present in nfκb2+/+ ILNs (Fig. 1⇓I), B cell areas in nfκb2−/− ILNs are small and poorly organized (Fig. 1⇓, I and J). Immunofluorescence staining of ILNs excised from 7-day-old mice with anti-CD45.2 Abs showed an almost complete absence of bone marrow-derived cells in organs from nfκb2−/− mice compared with nfκb2+/+ littermates (Fig. 1⇓, K and L). Taken together, these results show a paucity of T and B cells in ILNs of both adult and perinatal nfκb2−/− mice. A higher percentage of CD4+CD3− cells was observed in ILNs of nfκb2−/− neonates compared with nfκb2+/+ littermates (Fig. 2⇓A). Inducer cells from nfκb2+/+ and nfκb2−/− mice expressed similar levels of IL-7Rα on their surface (Fig. 2⇓B).

FIGURE 1.
FIGURE 1.

Impaired LN development in nfκb2−/− mice. A, ILNs from 8-wk-old nfκb2+/+ and nfκb2−/− littermate mice were isolated and analyzed on a dissection microscope (×4). Note the small size of the nfκb2−/− ILN compared with its WT littermate. B, nfκb2−/− ILNs have very low numbers of cells compared with WT littermates. Single-cell suspensions from nfκb2+/+ (▪) and nfκb2−/− mice (▦) ILNs were counted and analyzed by FACS for CD4+ T cells and B220+ B cells. Results shown correspond to the mean and SEM from two separate experiments using one ILN from six mice of each genotype per assay. Statistical analyses were performed by Mann-Whitney two-tailed test and showed very significant differences (p < 0.0043) between the two populations analyzed. Similar results were obtained in four independent experiments. C and D, FACS analyses of LN cells from nfκb2+/+ (C) and nfκb2−/− (D) littermate mice stained with anti-CD4-FITC and anti-CD8-PE mAbs. E and F, Histogram plots showing the percentage of B220+ B cells from ILNs of nfκb2+/+ (E) and nfκb2−/− (F) littermate mice. G–J, Immunostaining of representative sections of ILNs from adult nfκb2+/+ (G and I) and nfκb2−/− (H and J) littermates stained with anti-IgD (brown) for B cells and anti-CD3 (blue) for T cells. The rectangles highlight the areas that are magnified in I and J. Full circles in G and H indicate paracortical T cell areas. Arrow on G indicates B cells surrounding an HEV. Arrowheads in H indicate B cells present in the paracortex of the ILN of nfκb2−/− mice. Note the reduced number of B cells present in the nfκb2−/− ILN. K and L, Fluorescence micrograph of representative sections of ILNs from 7-day-old nfκb2+/+ (K) and nfκb2−/− (L) mice stained with anti-CD45.2 (green) and anti-PNAd (red). Note the low numbers of CD45.2+ bone marrow-derived cells present in the section of the nfκb2−/− ILN. Magnification ×20 (G and H) and ×40 (I–L).

FIGURE 2.
FIGURE 2.

Inducer cells are present in ILN of nfκb2−/− mice. A, FACS analyses of single cell suspensions from ILNs of 2-day-old WT and nfκb2−/− mice stained with anti-CD3-FITC/anti-CD4-PE. A higher percentage of CD4+/CD3− cells was observed in ILN of nfκb2−/− mice. B, Expression of IL-7Rα on CD4+/CD3− inducer cells is similar in WT and nfκb2−/− mice. Vertical lines in the histograms represent levels of background staining controls. Gated inducer cells were stained for IL-7Rα and analyzed.

LN organ cultures show a defect in nfκb2−/− stromal cells

To analyze whether the paucity of lymphocytes in nfκb2−/− LNs is due to intrinsic defects in lymphocytes or to defects in the stromal compartment, we established an organ culture system (LN organ culture) to study LN colonization under defined in vitro conditions (Fig. 3⇓A). Single-positive CD4+ thymocytes from nfκb2+/+ and nfκb2−/− mice were labeled with fluorescent tracker dyes. Equal numbers of cells were then placed in hanging drop cultures with alymphoid LNs of P2 wild-type (WT) or rag1−/− mice, and cell migration was assessed after 24 h by FACS. These assays showed that similar numbers of nfκb2−/− and nfκb2+/+ CD4+8− cells had migrated into WT LNs (Fig. 3⇓, A and B, and data not shown). In contrast, migration of cells to nfκb2−/− ILNs was reduced as compared with WT ILNs, regardless of whether the CD4+ cells were WT or nfκb2−/− (Fig. 3⇓B). Taken together, these results suggest that the reduced cellularity of ILNs in nfκb2−/− mice is due to a defect in stromal and/or endothelial cells rather than being intrinsic to lymphoid cells. However, the reduced size of nfκb2−/− ILNs compared with WT ILNs implies that fewer niches might be available to be colonized by lymphocytes, and thus results in small number of CD4+ cells migrating to the organs. Our studies show that nfκb2−/− CD4+ cells are capable of competing effectively with WT cells in their migration to LNs.

FIGURE 3.
FIGURE 3.

A defect in the stromal compartment of the LNs of nfκb2−/− mice is responsible for the reduced colonization of lymphocytes to these organs. A, LN organ culture assay. CD4+ cells labeled with Thy-1.2+ FITC (arrowheads) had migrated into an alymphoid P2 rag1−/− ILN after 24 h in culture. B, nfκb2−/− ILNs are colonized by low numbers of CD4+ thymocytes compared with nfκb2+/+ ILNs. CD4+CD8− thymocytes from littermate mice were labeled, as described in Materials and Methods, and used in LN organ culture assays on nfκb2+/+ and nfκb2−/− LNs. The organs were disaggregated, and cells were counted and analyzed by FACS. Note that nfκb2−/− CD4+ cells were able to colonize nfκb2+/+ ILN and compete with nfκb2+/+ cells. C, Neonate ILNs grafted under the kidney capsule of adult mice are populated by host cells. Photomicrograph of a kidney of an adult nfκb2−/− mouse carrying a 2-wk-old graft nfκb2+/+ ILN under the capsule. D, Similar as C, but showing an nfκb2−/− ILN grafted on a nfκb2+/+ mouse. LN grafts in C and D are highlighted by a blue box. Yellow box in D highlights the kidney capsule shown at higher magnification in F. E, Comparison of inguinal and mesenteric LNs from the nfκb2−/− recipient mouse and the nfκb2+/+ ILN graft from C. F, Same as E, showing inguinal and mesenteric LNs from the nfκb2+/+ recipient mouse and the nfκb2−/− ILN graft from D (blue rectangle) and surrounding kidney capsule (yellow rectangle).

NF-κB2 function is required in stromal cells, but not in lymphocytes for LN colonization

We next tested whether nfκb2−/− LNs can be colonized by lymphocytes in vivo. ILNs from neonatal nfκb2−/− and WT littermates were grafted under the kidney capsule of WT and nfκb2−/− adult mice, respectively (Fig. 3⇑, C–F) (39, 40, 41). After 2 wk, kidneys were dissected, and the grafts were analyzed. Fig. 3⇑C (blue box) shows a WT ILN grafted in an nfκb2−/− adult mouse, while Fig. 3⇑D (blue box) shows an nfκb2−/− ILN grafted in a WT adult mouse. Although the WT ILNs recovered were consistently found to have grown in size, recovered nfκb2−/− ILNs remained small, and in some cases could not be distinguished from the kidney capsule (Fig. 3⇑, C–F). Moreover, nfκb2−/− LNs grafted into WT hosts were found to be devoid of viable lymphocytes (Fig. 4⇓C). In marked contrast, viable T and B cells were readily recovered from WT ILNs grafted into nfκb2−/− mice (Fig. 4⇓A).

FIGURE 4.
FIGURE 4.

Neonate WT ILNs grafted on nfκb2−/− mice were colonized by lymphocytes from the host. A, WT Ly-5.1 ILN grafted on nfκb2−/− Ly-5.2 adult mice was colonized by CD4+ and CD8+ T cells and CD19+ B cells from the host. B, Bone marrow-derived cells migrating into grafted LNs are of host origin. Single cell suspensions from ILNs of nfκb2−/− (CD45.2) recipient mice and WT (CD45.1) donor grafts were analyzed by FACS for the expression of the CD45.1/CD45.2 alleles to identify the origin of cells migrating to the graft. As shown here, >90% of the cells are of host origin. C, nfκb2−/− ILN grafted on nfκb2+/+ adult mice was not colonized by lymphocytes from the host and contains dead cells. D–F, Immunofluorescence staining of an ILN section from the nfκb2−/− recipient mouse from A and B (D), nfκb2+/+ recipient mouse from C (E), and nfκb2+/+ ILN graft from A (F) for B220+ B cells (green) and CD3+ T cells (red). Note the small and loosely pack B cell area in the nfκb2−/− ILN (D) compared with the same area on the nfκb2+/+ graft ILN (F) and the nfκb2+/+ ILN (E).

Importantly, when we performed similar grafting experiments in a CD45.1/CD45.2 congenic system, we could clearly demonstrate that the vast majority (in all cases 90% or greater) of the CD4+ and CD8+, as well as the CD19+ B cells present within WT (CD45.1) ILN grafts, were of recipient nfκb2−/− CD45.2 origin, and not from cells pre-existing within the graft (Fig. 4⇑B).

Analysis of the distribution and organization of lymphocytes in the WT ILNs grafted into nfκb2−/− adults showed well-organized B and T cell areas (Fig. 4⇑F), which were indistinguishable from adult WT ILNs (Fig. 4⇑E). In contrast, small and disorganized B cell areas were present in the recipient nfκb2−/− adults’ own ILNs (Fig. 4⇑D). Therefore, when a WT LN microenvironment is provided, nfκb2−/− lymphocytes are capable of effective transendothelial migration into LNs and form compartmentalized B and T cell areas.

Expression of cell adhesion molecules is reduced in nfκb2−/− inguinal LNs

Analysis of the expression of cell adhesion molecules and receptors involved in lymphocyte migration into LNs, such as MAdCAM-1 and PNAd, in nfκb2+/+ and nfκb2−/− ILNs at different stages showed that these molecules were expressed at similar levels in both mice at P3 (data not shown). However, by P7, both PNAd and MAdCAM-1 were markedly reduced in the nfκb2−/− mice (Fig. 5⇓, A and B, and data not shown). Similarly, sections of 14- and 25-day-old nfκb2−/− ILNs showed low levels of expression of PNAd (Fig. 5⇓, C–F). Interestingly, ILNs from adult nfκb2−/− mice displayed a pattern of PNAd staining that resembles that of P7 nfκb2+/+ ILNs (Figs. 5⇓, G and H, and 6⇓, B and D). Close examination of PNAd expression in nfκb2−/− ILNs indicated a delay in the development and organization of the vessels, as shown by the few PNAd+ HEVs compared with the complexity of the HEV network in nfκb2+/+ ILNs and by the poorly organized PNAd+ and tomato lectin+ cells in ILNs of adult nfκb2−/− mice (Figs. 5⇓ and 6⇓, A and B). The calibre of the HEVs in nfκb2−/− ILNs was consistently reduced compared with nfκb2+/+ HEVs (Fig. 5⇓, A–H).

FIGURE 5.
FIGURE 5.

Reduced expression of PNAd in LNs of nfκb2−/− mice. Immunostaining of LN sections of nfκb2+/+ mice (A, C, E, and G) and nfκb2−/− mice (B, D, F, and H) for IgD+ B cells (brown) and the MECA79 Ab (blue), which recognizes a sugar moiety present in different PNAd such as CD34 and GlyCAM-1. A and B correspond to 7-day-old mice, C and D to 14-day-old mice, E and F to 25-day-old mice, and G and H to 3-mo-old mice. Arrowheads indicate PNAd+ HEVs, and the stars indicate B cell areas.

FIGURE 6.
FIGURE 6.

PNAd and tomato lectin staining in adult nfκb2−/− ILNs. Immunofluorescence staining of ILN sections of adult nfκb2+/+ mice (A), nfκb2−/− mice (B), a 2-wk-old nfκb2+/+ ILN grafted into a nfκb2−/− mouse (C), and a neonate nfκb2+/+ ILN (D) with MECA79 mAb (red) and tomato lectin (green). Note the PNAd luminal (star) and the tomato lectin abluminal staining (arrowhead) in nfκb2+/+ ILN (A) and the nfκb2+/+ graft (C) and the poorly organized HEV structure in nfκb2−/− ILN (B), which resembles the staining seen in neonate nfκb2+/+ ILNs (D). E, Sq reverse transcription and PCR amplification (Sq RT-PCR) of cell adhesion molecules GlyCAM-1, CD34, VCAM-1, and β actin as loading control. RNA was isolated from 14-day-old ILNs from nfκb2+/+ and nfκb2−/− mice, used to synthesize cDNA, and subjected to PCR amplification, as described in Materials and Methods. Lanes 1 and 7 correspond to 24 PCR cycles; lanes 2 and 8 to 28 cycles; lanes 3 and 9 to 32 cycles; lanes 4 and 10 to 36 cycles; lanes 5 and 11 to 40 cycles; and lanes 6 and 12 to 44 cycles. Note the marked reduction of expression of CD34 and GlyCAM-1 in the samples from nfκb2−/− mice. F, Sq RT-PCR of cell adhesion molecules in adult ILNs from nfκb2+/+ and nfκb2−/− mice. The expression of β actin, GlyCAM-1, CD34, and VCAM-1 was analyzed, as in E. Note the lower expression of CD34 and GlyCAM-1 in the samples from nfκb2−/− mice. Similar levels of VCAM-1 expression are shown in 14-day-old and adult tissues of nfκb2+/+ and nfκb2−/− mice.

Sq RT-PCR analysis confirmed our results in that mRNA levels of madcam-1 and the PNAd, glycam-1 and cd34, were markedly reduced in 14-day-old and adult nfκb2−/− ILNs compared with the nfκb2+/+ ILNs (Fig. 6⇑, E and F). Furthermore, the expression of the HEV-restricted sulfotransferase high endothelial cell-GlcNAc6 sulfotransferase (HEC-GlcNAc6ST) is strikingly decreased in nfκb2−/− ILNs (42, 43). Lymphocytes from the nfκb2−/− mice expressed similar levels of the PNAd receptor, L-selectin, and LFA-1 as their WT littermates (data not shown). These results clearly show that NF-κB2 is necessary for the normal expression of MAdCAM-1, glycosylation-dependent cell adhesion molecule (GlyCAM-1), and CD34.

NF-κB2 is necessary for the expression of CXCL13/B lymphocyte chemoattractant, CCL21/secondary lymphoid tissue chemokine, and CCL19/EBV-induced moleldule 1 ligand chemokine

The reduced number of lymphocytes recruited to nfκb2−/− ILNs prompted us to analyze the expression of the chemokines that facilitate lymphocyte transmigration of HEVs in LNs of adult mice (9, 10). Immunostaining of CXCL13 in ILNs showed a marked reduction at day 7–14 in nfκb2−/− mice, and it was undetectable in older mice compared with WT littermates (Fig. 7⇓A, and data not shown). Similarly, CD45− stromal cells from 3-day-old nfκb2−/− ILNs had noticeably lower mRNA levels of cxcl13, ccl21, and ccl19 compared with nfκb2+/+ littermates (Fig. 7⇓B). The impaired expression of CXCL13 and CCL21 was further confirmed in 14-day-old nfκb2−/− ILNs (Fig. 7⇓C). These results indicate a defect in stromal and endothelial cells in the absence of NF-κB2. Normal levels of cxcl12 and eotaxin expression were observed in nfκb2−/− ILNs (Fig. 7⇓, B and C). Sq RT-PCR analysis of cxcr5, ccr7, and cxcr4 showed no significant differences in the levels of these receptors in lymphocytes of nfκb2−/− and nfκb2+/+ mice (data not shown) (31).

FIGURE 7.
FIGURE 7.

Low expression of homing chemokines in LNs of nfκb2−/− mice. A, Immunostaining of ILN sections of adult nfκb2+/+ and nfκb2−/− mice with anti-CXCL13 antiserum (blue) and anti-IgD mAb (brown). Arrowheads indicate the presence of CXCL13 staining in endothelium of nfκb2+/+ mice. Stars indicate B cell areas. Note the absence of CXCL13 staining in nfκb2−/− mice and the low number of B cells. B, Sq RT-PCR of the chemokines CCL19, CCL21, CXCL13, CXCL12, eotaxin, and β actin as loading control. RNA was isolated from CD45− stromal cells from ILNs of 3-day-old nfκb2+/+ and nfκb2−/− mice. PCR amplification was performed as in Fig. 6⇑E. Note the marked decreased expression of CXCL13, CCL21, and CCL19 in the samples from nfκb2−/− mice. C, Sq RT-PCR of chemokine genes in 14-day-old ILNs from nfκb2+/+ and nfκb2−/− mice. Chemokine expression was analyzed, as in B.

Discussion

LN development involves interactions between bone marrow-derived inducer cells and stromal organizer cells to form cell clusters in the LN anlagen (7). Among the signals that mediate the cross talk between these cells as well as between lymphocytes and stromal cells in mature LNs, LTα1β2 binding to LTβR activates the classical and the alternative NF-κB pathways in stromal cells, resulting in nuclear translocation of p50/RelA and p52/RelB, respectively. In this study, we set forth to investigate the function of NF-κB2 p52 during LN development and show that p52 is cell autonomously required on LN stromal cells for the development of these organs.

Our study shows that in nfκb2−/− mice, ILNs are rudimentary, and have paucity of B cells compared with their WT counterparts. We established LN organ cultures and neonatal LN grafting assays to demonstrate that nfκb2−/− lymphocytes are fully capable of migrating into WT ILNs, and the BAFF-R/BR3 signaling defect in nfκb2−/− B cells does not play an important role in this process (44). Transfer of whole LN microenvironment overcomes the defect in ILN development in nfκb2−/− mice and shows that NF-κB2 has an important function during lymphocyte homing to LN.

Transendothelial migration of lymphocytes to peripheral LNs occurs through an adhesion cascade in several steps: 1) rolling via L-selectin+ lymphocytes interacting with PNAd+ endothelium; 2) lymphocyte activation by chemokine receptors; 3) sticking via LFA-1, diapedesis, and homing to the LN (45). The interaction between L-selectin+ lymphocytes and PNAd in HEVs is essential for lymphocyte traffic to PLNs, for l-selectin−/− mice have a dramatic reduction in size and cellularity of peripheral LNs due to reduced influx of lymphocytes via HEVs (46). This phenotype is similar to what we observed in nfκb2−/− ILNs, but in the latter is due to NF-κB2 regulating the expression of the L-selectin ligands GlyCAM-1 and CD34 and the sulfotransferase HEC-GlcNAc6ST, required for lumenal expression of the formers. Importantly, the endothelial vessels are less organized in nfκb2−/− ILNs compared with WT ILNs, indicating that LTα1β2/LTβR/NF-κB2 signaling between lymphocytes and endothelial cells might be required for full development of the HEV network. Alternatively, NF-κB2 might regulate the expression of signals from the stroma to the endothelium that stimulate HEV organization and growth. The factors that mediate stromal cell-endothelial cell communication are not well characterized. LTβR is expressed on human HEVs (12). Lack of appropriate reagents had precluded LTβR identification in mouse HEVs.

Overexpression of LTα3 and LTα1β2 or chemokines CXCL13 and CCL21 in mouse pancreas induces the development of ectopic lymphoid tissue, emphasizing the connection between chronic inflammation and lymphoid tissue organogenesis (47, 48, 49, 50, 51, 52). Congruently with our results, overexpression of LTα1β2, but no LTα3, specifically induces the expression of PNAd and HEC-GlcNAc6ST (51, 52).

In contrast to previous work showing that NF-κB2 has a role in posttranscriptional regulation of VCAM-1, the mRNA and protein levels of VCAM-1 showed no differences between nfκb2−/− and WT ILNs (Fig. 5⇑, E and F) (data not shown), suggesting that NF-κB2-independent signals might regulate the expression of this molecule in vivo (20).

NF-κB2 regulates the expression of the homing chemokines CXCL13, CCL21, and CCL19. However, the phenotype of the nfκb2−/− mice is milder than the cxcl13−/− or cxcr5−/− mice, for the latter develop only cervical, facial, and mesenteric LNs (11, 53). In contrast, plt/plt mice only occasionally lack a LN (3). CXCL13 acts as a retention factor for lymphocytes, suggesting that the reduced expression of this chemokine in nfκb2−/− mice could lead to difficulties in retaining B cells within the LN microenvironment and making them unable to migrate to the LN cortex and organize in B cell follicles (11, 53). As previously indicated, T cells express significantly higher levels of CCR7 than B cells and respond better to low concentration of CCL21, thus explaining the low numbers of B cells present in nfκb2−/− ILNs (9, 54).

In vivo studies blocking LTβR signaling during mouse embryogenesis have shown that the development of peripheral LNs occurs along the anterior-posterior axis of the body (55). ILNs begin to develop at embryonic day 16 (E16), followed by popliteal LNs at E17-E18. PPs develop between E16 and P1, and nasal-associated lymphoid tissue (NALT) from P0.5 to P7 (7). The results presented in this work indicate a function for NF-κB2 in lymphocyte homing to mature LNs. However, the fact that nfκb2−/− mice lack popliteal LNs, together with recent reports showing that these mice also lack PPs and have very small NALT, indicate that all the secondary lymphoid tissues that develop around E16 onward are markedly affected, demonstrating an important function for NF-κB2 at this developmental stage (7, 25, 27, 56).

Congruently with our results, mice overexpressing NF-κB2/p52, such as p100−/− mice, develop very large LNs (57). Collectively, our results demonstrate that NF-κB2 p52, together with its transcriptional partners RelB and RelA, and the kinases NIK and IKKα, has an important function in the LTβR-driven gene expression program regulating cell adhesion molecules and chemokines in stromal and endothelial cells required for lymphocyte trafficking and recruitment to LNs (Fig. 8⇓) (30, 31, 56).

FIGURE 8.
FIGURE 8.

Model of action of p52-containing complexes in stromal cells. p52/RelB and p52/RelA regulate the expression of CXCL13, CCL21, and CCL19, and the cell adhesion molecules MAdCAM-1, CD34, and GlyCAM-1. See text.

The analysis of LN development in different nfκb-deficient mouse strains shows that absence of NF-κB1 p105/p50 results in minimal changes in LN structure. Lack of RelA results in absence of LNs and PPs due to defects in stromal cells (33). Similarly, relb−/− neonate mice develop rudimentary LNs and lack PPs, and lymphocyte homing to NALT is impaired (25, 27). Thus, a hierarchy of NF-κB complexes necessary for LN organogenesis can be drawn with RelA- and RelB-containing complexes being essential and p52/RelA-p52/RelB also being necessary, although to a lesser extent. In the absence of NF-κB2/p52, NF-κB1 p50/RelA and p50/RelB heterodimers might partially compensate the absence of the former. A functional redundancy has been previously shown between p50 and p52, while RelA and RelB are unable to substitute each other, indicating that they regulate gene expression with high specificity (58, 59).

Our current model of LN development in nfκb2−/− mice indicates that colonization of LN anlagen by inducer cells occurs normally. However, the cross talk between inducer and organizer cells through LTβR results in activation of the classical NF-κB pathway only, with a subsequent low expression of adhesion molecules and chemokines. Therefore, the time required to reach the critical mass of bone marrow-derived cells into the LN anlagen is longer in nfκb2−/− embryos than in WT. We believe that a critical number of LTα1β2+ cells needs to be recruited to LNs in order for these organs to develop and organize the full structure. Consequently, the LNs that develop late during embryogenesis will be more affected than the ones that develop early. In addition, low influx and retention of lymphocytes to nfκb2−/− ILNs result in either very small or rudimentary organs. Analysis of different LNs in nfκb2−/− mice shows that most of these organs have a reduced size and cellularity, contain low numbers of B cells, but overall are not affected as ILNs.

Our studies emphasize the role of the stroma and the endothelium in the formation of ectopic lymphoid tissue in chronic inflammatory diseases such as rheumatoid arthritis, thyroid autoimmune disease, and Sjogren’s syndrome (60, 61, 62, 63).

Acknowledgments

We are indebted to Sonia Parnell and Dee McLoughlin for technical support, and Ravinder Suniara for invaluable help during this work. We are grateful to Anne-Gaelle Borycki for advice and helpful comments on this manuscript.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 The work in J.C.’s laboratory was supported by the Medical Research Council Centre for Immune Regulation, the University of Birmingham Medical School, Celltech Group (Berkshire, U.K.), and The Royal Society. A Medical Research Council Programme Grant supported the work of E.J. and G.A. A.E. was supported by a Medical Research Council Career Development Award. D.C., R.J., and A.W. were supported by Medical Research Council studentships. Additional support to R.J. was provided by Celltech Group.

  • ↵2 Address correspondence and reprint requests to Dr. Jorge Caamaño, Medical Research Council Centre for Immune Regulation, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT U.K. E-mail address: J.Caamano{at}bham.ac.uk

  • ↵3 Abbreviations used in this paper: LN, lymph node; E, embryonic day; GlyCAM-1, glycosylation-dependent cell adhesion molecule; HEC-GlcNAc6ST, high endothelial cell-GlcNAc6 sulfotransferase; HEV, high endothelial venule; IKK, IκB kinase; ILN, inguinal LN; LT, lymphotoxin; MAdCAM-1, mucosal addressin cell adhesion molecule-1; NALT, nasal-associated lymphoid tissue; NIK, NF-κB-inducing kinase; P, postnatal day; PNAd, peripheral node addressin; PP, Peyer’s patch; Sq, semiquantitative; TRANCE, TNF-related activation-induced cytokine; WT, wild type.

  • Received December 11, 2003.
  • Accepted June 3, 2004.

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