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B2–/– Mice Causes Impaired Lymph Node Development and Lymphocyte Recruitment1
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* Department of Anatomy,
Division of Infection and Immunity,
Medical Research Council Centre for Immune Regulation, and
Cancer Research United Kingdom Institute for Cancer Studies, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom
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Abstract |
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B 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-B 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-B family member NF-B2 in lymph node development and lymphocyte recruitment. Inguinal lymph nodes in nfb2–/– 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 nfb2–/– 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-B2 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-B2 in organization of lymph node endothelium. |
Introduction |
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Among the early events that give rise to LNs during embryonic development is the transendothelial migration of CD45+CD4+CD3–IL-7R
+4
7+ 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 12 (LT12), which upon binding to LTR 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 LT12/LTR in the latter (11).
Cross talk interactions between hemopoietic and stromal cells during LN development are mediated by LT12/LTR, TNF-related activation-induced cytokine (TRANCE)/TRANCE-R, TNF-/TNF-RI, and CXCL13/CXCR5. Gene knockout experiments have revealed that lt–/– and ltr–/– mice lack all LNs and PPs, while lt–/– mice have only mucosal LNs (12, 13). Likewise, trance–/– mice present with rudimentary LNs containing a reduced number of inducer cells (5). Signaling through LT12/LTR and, to a lesser degree, LT3/TNFR-I cooperates in the development of LNs and PPs (13, 14, 15).
Importantly, LTR, TRANCE-R, and TNF-RI signaling pathways converge in activation of the NF-B family of transcription factors. The Rel/NF-B 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-B proteins: NF-B1 (the DNA-binding subunit p50 and its precursor p105), NF-B2 (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 IBs. Activation of NF-B occurs via at least two distinct pathways (19). Signaling through the TNF-RI and other proinflammatory cytokine receptors activates the classical NF-B pathway through the IB kinase (IKK) signalosome complex, containing the IB kinases and (IKK, IKK), and a regulatory subunit, IKK
/NF-B essential modulator, induces phosphorylation and subsequent degradation of IB facilitating nuclear translocation of p50/RelA dimers. In contrast, LTR signals through both the classical and the alternative NF-B pathway that through the NF-B-inducing kinase (NIK) and an IKK complex induces the processing of NF-B2 p100 to p52 and the nuclear translocation of p52/RelB (20, 21, 22, 23, 24, 25, 26).
The NF-B proteins have specific functions during secondary lymphoid tissue organogenesis (27). We and others have shown that nfb2–/– 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-IB 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 IKKaa 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-B2 during LN development and lymphocyte recruitment to LNs. We show that nfb2–/– 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-B2 is required cell autonomously in bone marrow-derived or stromal cells during LN organization. We find that nfb2–/– lymphocytes are able to populate wild-type ILNs, whereas nfb2+/+ lymphocytes are dramatically impaired in their migration into nfb2–/– ILNs due to an intrinsic defect in the stromal cells of these organs. Our results demonstrate that NF-B2 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.
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Materials and Methods |
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The generation of nfb2–/– 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). Nfb2–/– CD4+ cells were labeled with CFSE, while the nfb2+/+ CD4+ cells were stained with PKH 2.6 (Sigma-Aldrich, St. Louis, MO). ILNs were excised from P2 nfb2+/+ and nfb2–/– mice and cultured in the presence of 1 x 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-7R, 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 nfb2+/+ and nfb2–/– mice and grafted by surgical procedure under the kidney capsule of 6- to 8-wk-old Ly-5.1 or Ly-5.2 C57BL6J nfb2+/+ or Ly-5.2 C57BL6J nfb2–/– mice. Grafts and host ILNs were extracted after 2 wk and analyzed by FACS analysis or immunostaining.
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Results |
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b2–/– mice
During the course of previous studies on nfb2–/– mice, we observed that peripheral LNs, in particular inguinal and popliteal LNs of adult mice, were dramatically reduced in size compared with LNs of nfb2+/+ littermates (Fig. 1A). This fact prompted us to pursue a thorough analysis of these organs to understand the cause of this impairment. Further observation demonstrated that adult nfb2–/– mice lack popliteal LNs. In addition, 40% of the adult nfb2–/– 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 nfb2–/– 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 nfb2–/– mice compared with nfb2+/+ littermates (Fig. 1, B–D): the mean of CD4+ T cells is 6-fold lower in nfb2–/– ILNs (nfb2+/+, 8.49 x 105 cells SEM 1.39 x 105; nfb2–/–, 1.38 x 105 cells SEM 2.53 x 104 (p < 0.0043)). Moreover, the number of B220+ B cells was found to be reduced 12-fold compared with nfb2+/+ ILNs (nfb2+/+, 2.78 x 105 cells SEM 5.7 x 104; nfb2–/–, 2.24 x 104 cells SEM 3.8 x 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 nfb2–/– mice showed an increase in the frequency of cells undergoing apoptosis, compared with nfb2+/+ mice (nfb2+/+, 45.5% SEM 2.3; nfb2–/–, 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 nfb2+/+ mice (Fig. 1, G and I) and nfb2–/– (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 nfb2–/– B cells were scattered among CD3+ T cells in the paracortex of the LNs (arrowheads, Fig. 1H). Large numbers of B cells were seen surrounding large HEVs in nfb2+/+ ILNs, but only few B cells were observed around small-sized HEVs in nfb2–/– 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 nfb2+/+ ILNs (Fig. 1I), B cell areas in nfb2–/– 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 nfb2–/– mice compared with nfb2+/+ 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 nfb2–/– mice. A higher percentage of CD4+CD3– cells was observed in ILNs of nfb2–/– neonates compared with nfb2+/+ littermates (Fig. 2A). Inducer cells from nfb2+/+ and nfb2–/– mice expressed similar levels of IL-7R on their surface (Fig. 2B).
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b2–/– stromal cells
To analyze whether the paucity of lymphocytes in nfb2–/– 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. 3A). Single-positive CD4+ thymocytes from nfb2+/+ and nfb2–/– 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 nfb2–/– and nfb2+/+ CD4+8– cells had migrated into WT LNs (Fig. 3, A and B, and data not shown). In contrast, migration of cells to nfb2–/– ILNs was reduced as compared with WT ILNs, regardless of whether the CD4+ cells were WT or nfb2–/– (Fig. 3B). Taken together, these results suggest that the reduced cellularity of ILNs in nfb2–/– mice is due to a defect in stromal and/or endothelial cells rather than being intrinsic to lymphoid cells. However, the reduced size of nfb2–/– 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 nfb2–/– CD4+ cells are capable of competing effectively with WT cells in their migration to LNs.
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B2 function is required in stromal cells, but not in lymphocytes for LN colonization
We next tested whether nfb2–/– LNs can be colonized by lymphocytes in vivo. ILNs from neonatal nfb2–/– and WT littermates were grafted under the kidney capsule of WT and nfb2–/– adult mice, respectively (Fig. 3, C–F) (39, 40, 41). After 2 wk, kidneys were dissected, and the grafts were analyzed. Fig. 3C (blue box) shows a WT ILN grafted in an nfb2–/– adult mouse, while Fig. 3D (blue box) shows an nfb2–/– ILN grafted in a WT adult mouse. Although the WT ILNs recovered were consistently found to have grown in size, recovered nfb2–/– ILNs remained small, and in some cases could not be distinguished from the kidney capsule (Fig. 3, C–F). Moreover, nfb2–/– LNs grafted into WT hosts were found to be devoid of viable lymphocytes (Fig. 4C). In marked contrast, viable T and B cells were readily recovered from WT ILNs grafted into nfb2–/– mice (Fig. 4A).
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b2–/– CD45.2 origin, and not from cells pre-existing within the graft (Fig. 4B).
Analysis of the distribution and organization of lymphocytes in the WT ILNs grafted into nfb2–/– adults showed well-organized B and T cell areas (Fig. 4F), which were indistinguishable from adult WT ILNs (Fig. 4E). In contrast, small and disorganized B cell areas were present in the recipient nfb2–/– adults’ own ILNs (Fig. 4D). Therefore, when a WT LN microenvironment is provided, nfb2–/– 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 nfb2–/– 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 nfb2+/+ and nfb2–/– 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 nfb2–/– mice (Fig. 5, A and B, and data not shown). Similarly, sections of 14- and 25-day-old nfb2–/– ILNs showed low levels of expression of PNAd (Fig. 5, C–F). Interestingly, ILNs from adult nfb2–/– mice displayed a pattern of PNAd staining that resembles that of P7 nfb2+/+ ILNs (Figs. 5, G and H, and 6, B and D). Close examination of PNAd expression in nfb2–/– 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 nfb2+/+ ILNs and by the poorly organized PNAd+ and tomato lectin+ cells in ILNs of adult nfb2–/– mice (Figs. 5 and 6, A and B). The calibre of the HEVs in nfb2–/– ILNs was consistently reduced compared with nfb2+/+ HEVs (Fig. 5, A–H).
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b2–/– ILNs compared with the nfb2+/+ 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 nfb2–/– ILNs (42, 43). Lymphocytes from the nfb2–/– 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-B2 is necessary for the normal expression of MAdCAM-1, glycosylation-dependent cell adhesion molecule (GlyCAM-1), and CD34.
NF-B2 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 nfb2–/– 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 nfb2–/– mice, and it was undetectable in older mice compared with WT littermates (Fig. 7A, and data not shown). Similarly, CD45– stromal cells from 3-day-old nfb2–/– ILNs had noticeably lower mRNA levels of cxcl13, ccl21, and ccl19 compared with nfb2+/+ littermates (Fig. 7B). The impaired expression of CXCL13 and CCL21 was further confirmed in 14-day-old nfb2–/– ILNs (Fig. 7C). These results indicate a defect in stromal and endothelial cells in the absence of NF-B2. Normal levels of cxcl12 and eotaxin expression were observed in nfb2–/– 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 nfb2–/– and nfb2+/+ mice (data not shown) (31).
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Discussion |
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12 binding to LTR activates the classical and the alternative NF-B 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-B2 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 nfb2–/– 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 nfb2–/– lymphocytes are fully capable of migrating into WT ILNs, and the BAFF-R/BR3 signaling defect in nfb2–/– B cells does not play an important role in this process (44). Transfer of whole LN microenvironment overcomes the defect in ILN development in nfb2–/– mice and shows that NF-B2 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 nfb2–/– ILNs, but in the latter is due to NF-B2 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 nfb2–/– ILNs compared with WT ILNs, indicating that LT12/LTR/NF-B2 signaling between lymphocytes and endothelial cells might be required for full development of the HEV network. Alternatively, NF-B2 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. LTR is expressed on human HEVs (12). Lack of appropriate reagents had precluded LTR identification in mouse HEVs.
Overexpression of LT3 and LT12 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 LT12, but no LT3, specifically induces the expression of PNAd and HEC-GlcNAc6ST (51, 52).
In contrast to previous work showing that NF-B2 has a role in posttranscriptional regulation of VCAM-1, the mRNA and protein levels of VCAM-1 showed no differences between nfb2–/– and WT ILNs (Fig. 5, E and F) (data not shown), suggesting that NF-B2-independent signals might regulate the expression of this molecule in vivo (20).
NF-B2 regulates the expression of the homing chemokines CXCL13, CCL21, and CCL19. However, the phenotype of the nfb2–/– 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 nfb2–/– 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 nfb2–/– ILNs (9, 54).
In vivo studies blocking LTR 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-B2 in lymphocyte homing to mature LNs. However, the fact that nfb2–/– 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-B2 at this developmental stage (7, 25, 27, 56).
Congruently with our results, mice overexpressing NF-B2/p52, such as p100–/– mice, develop very large LNs (57). Collectively, our results demonstrate that NF-B2 p52, together with its transcriptional partners RelB and RelA, and the kinases NIK and IKK, has an important function in the LTR-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).
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b-deficient mouse strains shows that absence of NF-B1 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-B 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-B2/p52, NF-B1 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 nfb2–/– mice indicates that colonization of LN anlagen by inducer cells occurs normally. However, the cross talk between inducer and organizer cells through LTR results in activation of the classical NF-B 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 nfb2–/– embryos than in WT. We believe that a critical number of LT12+ 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 nfb2–/– ILNs result in either very small or rudimentary organs. Analysis of different LNs in nfb2–/– 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).
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Acknowledgments |
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Footnotes |
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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, IB kinase; ILN, inguinal LN; LT, lymphotoxin; MAdCAM-1, mucosal addressin cell adhesion molecule-1; NALT, nasal-associated lymphoid tissue; NIK, NF-B-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 for publication December 11, 2003. Accepted for publication June 3, 2004.
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) and nf
) 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 nf
