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The Journal of Immunology, 2006, 176: 3995-4002.
Copyright © 2006 by The American Association of Immunologists

Essential Role of I{kappa}B Kinase {alpha} in Thymic Organogenesis Required for the Establishment of Self-Tolerance1

Dan Kinoshita*, Fumiko Hirota{ddagger}, Tsuneyasu Kaisho§, Michiyuki Kasai, Keisuke Izumi{dagger}, Yoshimi Bando{dagger}, Yasuhiro Mouri{ddagger}, Akemi Matsushima{ddagger}, Shino Niki{ddagger}, Hongwei Han{ddagger}, Kiyotaka Oshikawa{ddagger}, Noriyuki Kuroda{ddagger}, Masahiko Maegawa*, Minoru Irahara*, Kiyoshi Takeda||, Shizuo Akira# and Mitsuru Matsumoto2,{ddagger}

* Department of Obstetrics and Gynecology and {dagger} Department of Molecular and Environmental Pathology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; {ddagger} Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan; § Laboratory for Host Defense, Research Center for Allergy and Immunology, Kanagawa, Japan; Division of Bacterial and Blood Products, National Institute of Infectious Disease, Tokyo, Japan; || Division of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; and # Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
I{kappa}B kinase (IKK) {alpha} exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-{kappa}B activation pathway. The in vivo function of IKK{alpha}, however, still remains elusive. Because a natural strain of mice with mutant NF-{kappa}B-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKK{alpha} axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKK{alpha}-deficient mice. The thymic microenvironment that caused autoimmunity in an IKK{alpha}-dependent manner was associated with defective processing of NF-{kappa}B2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKK{alpha} in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The transcription factor NF-{kappa}B plays an important role in the regulation of innate immunity, stress responses, inflammation, and the inhibition of apoptosis (1, 2). The activity of NF-{kappa}B is tightly regulated through the I{kappa}B kinase (IKK)3 complex, which consists of two catalytic subunits (IKK{alpha} and IKKbeta) and a regulatory subunit (IKK{gamma}) (2). IKK{alpha} has been shown to be phosphorylated by NF-{kappa}B-inducing kinase (NIK) (3), which is structurally related to MEK kinase (4). Many aspects of the in vivo function of these key players have been elucidated by the use of both gene-targeted mice and natural mutant mice (2). The alymphoplasia (aly) strain of mice carries a natural mutation of the NIK gene (5, 6) in which a G855R substitution in the C terminus of the protein results in inability to bind to IKK{alpha} (7). We have demonstrated previously that a defective NIK-IKK{alpha} axis downstream of lymphotoxin (LT) betaR, a receptor essential for secondary lymphoid organogenesis (8), is responsible for the abnormal development of secondary lymphoid organs in aly mice (7, 9).

In addition to its essential role in secondary lymphoid organogenesis, we have demonstrated recently that NIK is required in the thymic stroma for the organization of the thymic microenvironment (10). Abnormal thymic organogenesis in the absence of normal NIK accounts for the autoimmune disease phenotype seen in aly mice, which is characterized by chronic inflammatory changes in several organs, including the liver, pancreas, salivary gland, and lacrimal gland (5, 10). Because breakdown of self-tolerance is considered to be the key event responsible for the autoimmune disease process, and establishment of self-tolerance primarily depends on physical contact between thymocytes and thymic stroma (11), characterization of the stromal elements involved may contribute to the development of a therapeutic approach to many autoimmune diseases.

Medullary thymic epithelial cells (mTECs) play pivotal roles in the cross talk between developing thymocytes and thymic stroma (12). Elimination of autoreactive T cells (negative selection) and/or production of immunoregulatory T cells (Tregs) are most likely mediated by a set of self Ags expressed on mTECs (13, 14). In fact, gene expression studies have demonstrated that mTECs are a specialized cell type in which promiscuous expression of a broad range of tissue-specific Ag (TSA) genes is an autonomous property (15). Studies on mice with autoimmune phenotypes resulting from an abnormal thymic microenvironment have provided useful insights into this mechanism. Autoimmune regulator (Aire)-deficient mice have mTECs with reduced expression of many, but not all, TSAs, but have apparently normal thymic structure (16, 17). These results suggest that Aire regulates transcription of TSAs within developed mTECs without influencing the development of these cells. In contrast, reduced expression of TSAs (and Aire) in the thymus from NIK mutant mice is associated with impaired development of mTECs (10); NIKaly/aly mice lack Ulex europaeus (UEA)-1+ mTECs and have reduced numbers of ER-TR5+ mTECs. Although these results are consistent with the idea that NIK affects TSA expression in the thymus through a developmental effect on mTECs, it is not clear whether NIK has any significant roles in the transcription of TSA genes within these cells, as suggested for Aire.

Initial studies of mice deficient in IKK{alpha} have unveiled an unexpected function of IKK{alpha} for the development of limbs and skin (18, 19). Subsequent studies have revealed a two-dimensional role for IKK{alpha}, which possesses both protein kinase-dependent and protein kinase-independent functions. It has been demonstrated that kinase activity is required for lymphoid organogenesis (7, 20), B cell development and function (21), and mammary gland development (22). In contrast, kinase-independent activity is required for epidermal keratinocyte differentiation and skeletal and craniofacial morphogenesis (23, 24). Perinatal death of IKK{alpha}–/– mice, however, has hampered a detailed analysis of the in vivo immunological function of IKK{alpha}. Given that NIKaly/aly mice have disorganized thymic structure together with an organ-specific autoimmune disease, we hypothesized that IKK{alpha} in the thymic stroma has similar roles to those of NIK. In the present study, we have examined this hypothesis and demonstrated that IKK{alpha} regulates thymic organogenesis and establishes self-tolerance primarily through a noncanonical NF-{kappa}B activation pathway with NIK, which requires the processing of NF-{kappa}B2 (i.e., production of p52 from its precursor p100) (25). With the use of isolated thymic epithelial cells (TECs) together with mTEC lines established from NIKaly/aly mice, we have also demonstrated that the NIK-IKK{alpha} axis regulates thymic expression of TSAs predominantly through the developmental process of mTECs, not through transcriptional control of TSA genes within developed mTECs. Thus, our results illustrate a novel function of IKK{alpha} in thymic stroma-dependent self-tolerance that cannot be compensated for by the related IKKbeta subunit.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/cA Jcl-{nu} mice (BALB/cnu/nu mice) and NIKaly/aly mice were purchased from CLEA Japan, and Rag2-deficient mice on BALB/c background were acquired from Taconic Farms. IKK{alpha}–/– mice were generated by gene targeting, as described previously (18). The mice were maintained under pathogen-free conditions and were handled in accordance with the Guidelines for Animal Experimentation of Tokushima University School of Medicine.

Thymus grafting

Thymus grafting was performed, as previously described (10). Briefly, thymic lobes were isolated from embryos at 14.5 days postcoitus and were cultured for 4 days on top of Nucleopore filters (Whatman) placed on RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, and 1.35 mM 2'-deoxyguanosine (2-DG; Sigma-Aldrich). Five pieces of thymic lobes were grafted under the renal capsule of BALB/cnu/nu mice. After 6–8 wk, reconstitution of peripheral T cells was determined by flow cytometric analysis (BD Biosciences) with anti-CD4 (clone GK1.5; BD Pharmingen) and anti-CD8 (clone 53-6.7; BD Pharmingen) mAbs, and then thymic chimeras were used for the analyses.

Western blotting

Proteins extracted from embryonic thymic lobes, prepared as described above, were analyzed with an ECL Western blotting detection system (Amersham Biosciences). Rabbit anti-peptide Abs directed against p52 (catalog no. sc-298) and RelB (catalog no. sc-226), mouse anti-lck mAb (catalog no. sc-433), and goat anti-actin Ab (catalog no. sc-1616) were all purchased from Santa Cruz Biotechnology.

Pathology

Formalin-fixed tissue sections were subjected to H&E staining, and two pathologists independently evaluated the histology without being informed of the detailed condition of the individual mouse. Histological changes were scored as 0 (no change), 1 (mild lymphoid cell infiltration), or 2 (marked lymphoid cell infiltration).

Establishment of TEC lines from NIKaly/aly mice

TEC lines were established from NIKaly/aly embryos at 14.5 days postcoitus, as previously described (26). These cells were maintained with gamma ray-irradiated (40 Gy) Swiss 3T3 cells as feeder cells in calcium-free MEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS, 3 mM L-glutamine, 50 µg/ml gentamicin, 50 µM 2-ME, and 1 µg/ml hydrocortisone (Sigma-Aldrich). NIKaly/aly mouse origin was confirmed by sequencing of the aly-type NIK gene (6, 7).

Immunohistochemistry

Immunohistochemical analysis of the grafted thymus was performed, as previously described (10). Briefly, frozen tissue sections were fixed in cold acetone and stained by first incubating them with ER-TR5 (27) and UEA-1-biotin (Vector Laboratories). After being washed, the sections were further incubated with Alexa 594-conjugated goat anti-rat IgG (Invitrogen Life Technologies) and Alexa 488-conjugated streptavidin (Invitrogen Life Technologies) for the immunofluorescence. For the detection of autoantibodies, serum from thymic chimeras was incubated with various organs obtained from Rag2-deficient mice. FITC-conjugated anti-mouse IgG Ab (Southern Biotechnology Associates) was used for the detection. Polyclonal anti-Aire Ab was produced by immunizing rabbits with peptides corresponding to the COOH-terminal portion of mouse Aire, and Alexa 488-conjugated donkey anti-rabbit IgG (Invitrogen Life Technologies) was used as a secondary Ab for detection. TEC lines established from NIKaly/aly embryos were seeded on coverslips and subjected to immunohistochemistry, as previously described (28). Anti-epithelial cell adhesion molecule (Ep-CAM) mAb (BD Biosciences) and anti-keratin-5 polyclonal Ab (Covance) were used for the staining. DNA staining was with 4'6-diamidino-2-phenylindole (Roche Applied Science).

NF-{kappa}B2 processing

TECs were stimulated with agonistic anti-LTbetaR mAb (clone AF.H6; provided by P. Rennert, Biogen Idec) (29) (5 µg/ml) or with agonistic anti-CD40 mAb (clone 3/23; Serotec) (5 µg/ml) for 8 h. Cytoplasmic and nuclear extracts were prepared from the cells, as described previously (30), and were subjected to Western blotting with rabbit anti-p52 Ab from Upstate Biotechnology (catalogue 06-413).

Thymic stroma preparation

Thymic stroma was prepared, as described previously (17). Briefly, thymic lobes were isolated from three to six mice for each group and cut into small pieces. The fragments were gently rotated in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies), 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME, hereafter referred to as R10, at 4°C for 30 min, and dispersed further with pipetting to remove the majority of thymocytes. The resulting thymic fragments were digested with 0.15 mg/ml collagenase IV (Sigma-Aldrich) and 10 U/ml DNase I (Roche Molecular Biochemicals) in RPMI 1640 at 37°C for 15 min. The supernatants that contained dissociated TECs were saved, whereas the remaining thymic fragments were further digested with collagenase IV and DNase I. This step was repeated twice, and the remaining thymic fragments were digested with collagenase IV, DNase I, and 0.1 mg/ml dispase I (Roche Applied Science) at 37°C for 30 min. The supernatants from this digest were combined with the supernatants from the collagenase digests, and the mixture was centrifuged for 5 min at 450 x g. The cells were suspended in PBS containing 5 mM EDTA and 0.5% FCS and kept on ice for 10 min. CD45– thymic stromal cells were then purified by depleting CD45+ cells with MACS CD45 microbeads (Miltenyi Biotec), according to the manufacturer’s instructions. The resulting preparations contained ~60% Ep-CAM+ cells and <10% thymocytes (i.e., CD4/CD8 single-positive and CD4/CD8 double-positive cells), as determined by flow cytometric analysis.

Real-time PCR and semiquantitative RT-PCR

Real-time PCR for quantification of TSA genes was conducted with cDNA prepared from RNAs extracted from whole thymus or from isolated TECs. The primers, the probes, and the reactions were those described previously (10, 17). Cathepsin S primers were 5'-GCCATTCCTCCTTCTTCTTCTACA-3' and 5'-CAAGAACACCATGATTCACATTGC-3', and the cathepsin S probe was 5'-FAM-AAGCGGTGTCTATGATGACCCCTCCTGTA-3' (31). Semiquantitative RT-PCR of TSA genes was conducted, as previously described (10, 17).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IKK{alpha} in the thymic stroma is required for self-tolerance

We have demonstrated recently that aly mice, a natural strain with mutant NIK, manifest autoimmunity resulting from disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma (10). Although the identity of the upstream receptor(s) controlling NIK-dependent thymic organogenesis has not been fully determined (see Discussion), we speculated that IKK{alpha} might function as a downstream kinase of NIK in this process. Because of the perinatal death of IKK{alpha}–/– mice (18, 19), we assessed thymic organogenesis and T cell development in IKK{alpha}–/– mice by using embryonic thymus; thymic lobes were isolated from control and IKK{alpha}–/– embryos at 14.5 days postcoitus and cultured for 4 days in vitro. Such thymic lobes supported maturation of thymocytes similarly in both control and IKK{alpha}–/– mice (Fig. 1), indicating a dispensable role of IKK{alpha} in both thymocytes and thymic stroma in their developmental cross talk. The dispensability of IKK{alpha} in thymocyte development assessed with this fetal thymus organ culture system is consistent with the observation of normal T cell development in chimeras in which IKK{alpha}–/– fetal liver cells were transferred into irradiated Rag2-deficient mice (21). Of importance, histological examination of those chimeras showed no signs of autoimmune disease (T. Kaisho, K. Izumi, and M. Matsumoto, unpublished observation), suggesting that IKK{alpha}-deficient T cells do not promote the development of autoimmune disease in a cell-autonomous manner. In contrast, we speculated that IKK{alpha} in thymic stroma might be essential for the establishment of self-tolerance, as demonstrated for NIK (10). To test this hypothesis, we generated thymic chimeras. The 2-DG-treated embryonic thymic lobes, which did not contain any live thymocytes as determined by flow cytometric analysis (see Fig. 1, left panels) and by Western blotting with anti-lck Ab (see Fig. 2, top panel), were prepared and then grafted under the renal capsule of BALB/cnu/nu mice. In this system, mature T cells derived from IKK{alpha}-sufficient recipient BALB/cnu/nu mouse bone marrow are produced de novo through interaction with the grafted thymic stroma. Grafting both control and IKK{alpha}–/– embryonic thymus induced T cell maturation in the periphery of BALB/cnu/nu mice to a similar extent: CD4+ T cells plus CD8+ T cells were 14.1 ± 5.3% in BALB/cnu/nu mice grafted with control thymus (n = 6) compared with 15.1 ± 7.6% in BALB/cnu/nu mice grafted with IKK{alpha}–/– thymus (n = 7). Remarkably, histological examination of IKK{alpha}–/– thymus-grafted mice, but not control thymus-grafted mice, revealed many lymphoid cell infiltrations in the liver, mainly in the portal area (Fig. 3, A and B), which is reminiscent of the autoimmune disease phenotype observed in NIKaly/aly mice. To see whether T cells developed in a thymic microenvironment without IKK{alpha} in those mice are autoreactive per se, we injected splenocytes obtained from BALB/cnu/nu mice grafted with IKK{alpha}-deficient thymus into another group of BALB/cnu/nu mice. We observed similar lymphoid cell infiltration in the liver of the recipient mice, whereas injection of splenocytes obtained from BALB/cnu/nu mice grafted with control thymus induced no such changes in the recipients (Fig. 3B). These results clearly indicate the significance of IKK{alpha} as a thymic stromal element required for the establishment of self-tolerance. Five of seven IKK{alpha}–/– thymus-grafted mice also showed lymphoid cell infiltrations in the pancreas (perivascular areas near islets), although these infiltrations were less marked than in the liver (D. Kinoshita, K. Izumi, and M. Matsumoto, unpublished observation).


Figure 1
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  		FIGURE 1. Unaltered thymocyte development in the absence of IKK{alpha} in fetal thymic organ culture. Thymic lobes isolated from embryos of both control (center panels) and IKK{alpha}–/– (right panels) mice at 14.5 days postcoitus supported maturation of thymocytes similarly in a 4-day organ culture in the absence of 2-DG. Flow cytometric analysis with forward scatter (FSC) and side scatter (SSC) (top panels), and with anti-CD4 and anti-CD8 mAbs (bottom panels). Thymic organ culture of control mice in the presence of 2-DG is shown as a negative control (left panels). One representative result from a total of two repeats is shown.

 

Figure 2
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  		FIGURE 2. IKK{alpha} regulates the processing of NF-{kappa}B2 in thymic stroma. Thymic lobes isolated from control (second lane) and IKK{alpha}–/– embryos (third lane), and cultured for 4 days in the presence of 2-DG contain no live thymocytes, as demonstrated by the lack of lck expression with Western blotting (top panel). The same blot was probed with anti-Rel protein Abs (two middle panels) and anti-actin Ab (bottom panel). p52 processing from the precursor p100 was impaired in thymic stroma from IKK{alpha}–/– mice. RelB expression was also reduced in IKK{alpha}–/– thymus. Total splenocytes (Spl) from wild-type mice were used as control (first lane). Intensities of the bands of p100 and p52 in each lane were measured with ImageJ software (National Institutes of Health), and the ratios between p52 and p100 are shown above the NF-{kappa}B2 Western blot. One representative result from a total of three repeats is shown.

 

Figure 3
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  		FIGURE 3. Requirement for IKK{alpha} in thymic stroma for the establishment of self-tolerance. A, BALB/cnu/nu mice grafted with IKK{alpha}–/– embryonic thymus (right panel), but not with control embryonic thymus (left panel), developed an autoimmune disease phenotype in the liver. Arrows indicate the lymphoid cell infiltrations. The scale bar corresponds to 100 µm in size. B, Many IKK{alpha}–/– thymus-grafted BALB/cnu/nu mice exhibited lymphoid cell infiltrations in the liver (•; left half panel). In contrast, these changes were scarcely observed in control thymus-grafted mice ({circ}). Injection of splenocytes obtained from BALB/cnu/nu mice grafted with IKK{alpha}-deficient thymus into another group of BALB/cnu/nu mice induced lymphoid cell infiltration in the liver of the recipient mice (•; right half panel), whereas injection of splenocytes obtained from BALB/cnu/nu mice grafted with control thymus induced no such changes in the recipient mice ({circ}). Histological changes in H&E-stained tissue sections were scored as 0 (no change), 1 (mild lymphoid cell infiltration), or 2 (marked lymphoid cell infiltration). One mark corresponds to one mouse analyzed. C, Serum from BALB/cnu/nu mice grafted with IKK{alpha}–/– thymus (right panels), but not with control thymus (left panels), contained IgG class autoantibodies against liver (Li; top panels), stomach (St; middle panels), and kidney (Ki; bottom panels) detected with immunofluorescence. Original magnification, x100.

 
Development of autoimmunity in athymic nude mice grafted with IKK{alpha}–/– thymus was further demonstrated by the production of autoantibodies against various organs. When the serum from BALB/cnu/nu mice grafted with IKK{alpha}–/– thymus was tested for reactivity against liver, six of seven showed IgG class autoantibodies as detected with immunofluorescence (Fig. 3C). In contrast, such activity was observed in only one of six control thymus-grafted mice, and this activity was only weak. Similarly, high incidences of autoantibodies against stomach (five of seven) and kidney (six of seven) were observed in IKK{alpha}–/– thymus-grafted mice, although on histological examination these organs appeared unaffected when assessed 6–8 wk after thymus graft (D. Kinoshita, K. Izumi, and M. Matsumoto, unpublished observation).

IKK{alpha} in the thymic stroma regulates Rel protein expression and thymic organogenesis

Given that IKK{alpha} plays an essential role in the thymic microenvironment that is required for the establishment of self-tolerance, we investigated the expression of Rel proteins from IKK{alpha}–/– thymic stroma by Western blotting. Thymic lobes used for this experiment were isolated from control and IKK{alpha}–/– embryos at 14.5 days postcoitus and treated with 2-DG to isolate only thymic stromal elements, as described above. Expression of p52 was significantly reduced in the thymic stroma from IKK{alpha}–/– mice compared with that from control mice, whereas p100, a precursor form of p52, was more abundant in IKK{alpha}–/– mice than in control mice (Fig. 2); the amount of p52 in thymic stroma from control mice was double that of p100, whereas the ratio of p52 to p100 was reversed in IKK{alpha}–/– mice. Thus, IKK{alpha}-dependent generation of p52 from p100 in thymic stroma might constitute a second NF-{kappa}B signaling pathway, as we originally observed in hemopoietic cells (32) and subsequently characterized for signals through LTbetaR (33), CD40 (34), and B cell activating factor of the TNF family receptor (35, 36). RelB expression in the thymic stroma was slightly reduced in IKK{alpha}–/– mice compared with that in control mice (Fig. 2), as observed in NIKaly/aly mice (10). These results suggest that the disturbed thymic microenvironment in IKK{alpha}–/– mice is associated with abnormal regulation of the NF-{kappa}B activation pathway in the thymic stroma in the absence of IKK{alpha}.

The essential roles of IKK{alpha} in thymic stroma were also confirmed by histological examination of the grafted thymus. Although embryonic thymus from control mice that had been grafted onto BALB/cnu/nu mice contained mTECs that bound with UEA-1, IKK{alpha}–/– embryonic thymus grafted onto BALB/cnu/nu mice did not have UEA-1+ cells (Fig. 4A). ER-TR5+ mTECs were sparse in IKK{alpha}–/– embryonic thymus grafted onto BALB/cnu/nu mice compared with control embryonic thymus grafted similarly (Fig. 4A). Abnormal development of mTECs in the absence of IKK{alpha} was also exemplified by the loss of Aire+ cells in IKK{alpha}–/– embryonic thymus grafted onto BALB/cnu/nu mice (Fig. 4B). A dramatic decrease in the number of Aire+ cells was also observed in adult untreated NIKaly/aly thymus (Fig. 4B). Because T cells with normal IKK{alpha} (derived from BALB/cnu/nu mice) cannot restore normal mTECs in the IKK{alpha}–/– thymus when the interaction between T cells and thymic stromal cells is initiated from the embryonic stage, the contribution of IKK{alpha} to thymic organogenesis seems to be stromal element autonomous. These results clearly indicate indispensable roles for IKK{alpha} as a stromal element in the thymic organogenesis that is required for the establishment of self-tolerance.


Figure 4
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  		FIGURE 4. IKK{alpha} is required for thymic organization. A, Embryonic thymus from IKK{alpha}–/– mice contained no UEA-1+ cells (bottom middle panel) and fewer ER-TR5+ medullary epithelial cells stained in red (bottom left panel) after grafting onto BALB/cnu/nu mice compared with that from control mice (top left panel). UEA-1+ cells from control embryonic thymus grafted onto BALB/cnu/nu mice were stained in green (top middle panel), and were merged with ER-TR5 staining (top right panel). B, Embryonic thymus from IKK{alpha}–/– mice grafted onto BALB/cnu/nu mice contained very few Aire+ cells (top right panel), as observed in adult untreated NIKaly/aly thymus (bottom right panel). Aire+ cells were observed in embryonic thymus from control mice grafted onto BALB/cnu/nu mice (top left panel) and adult untreated NIKaly/+ thymus (bottom left panel). Original magnification, x200. One representative result from a total of five repeats is shown.

 
Developmental effect of NIK for promiscuous gene expression in the thymus

Promiscuous gene expression of many TSAs in mTECs could play an essential role in the establishment of central tolerance (12). The autoimmunity developed in NIKaly/aly mice (10) and IKK{alpha}–/– mice, described above, might be associated with altered expression of self Ags in the thymus. In fact, NIKaly/aly thymus showed dramatically reduced transcription of many TSAs (10). We have examined whether thymic expression of TSAs is influenced by the absence of IKK{alpha} using embryonic thymus grafted onto BALB/cnu/nu mice; RNAs were extracted from the thymus 6 wk after grafting when the thymus was colonized with developing thymocytes derived from BALB/cnu/nu mouse bone marrow. By real-time PCR, salivary protein 1 (SP1), fatty acid-binding protein (FABP), C-reactive protein (CRP), and glutamic acid decarboxylase 67 (GAD67) were easily detected in grafted control thymus, whereas expression of SP1, FABP, and GAD67 was below the limit of detection in grafted IKK{alpha}–/– thymus. Although CRP was detected in grafted IKK{alpha}–/– thymus (N. Kuroda and M. Matsumoto, unpublished observation), its expression was reduced; the value for CRP/hypoxanthine phosphoribosyltransferase (HPRT) from control thymus was 1.61, and that for CRP/HPRT from IKK{alpha}–/– thymus was 0.32.

Because we used RNAs extracted from total thymus instead of isolated mTECs in both previous experiments with NIKaly/aly mice (10) and experiments with IKK{alpha}–/– thymic chimeras described above, it is not clear whether reduced expression of TSAs was due to the reduced number of mTECs expressing TSAs (15) or to the lack of NIK-IKK{alpha}-dependent transcriptional control of TSA genes. To test these possibilities, we harvested TECs (which contain both cortical and medullary components) from adult NIKaly/aly mice and examined the expression of TSAs together with cathepsin S (CAT-S), which is highly expressed by mTECs in the thymic stroma (31). Consistent with immunohistochemical evaluation demonstrating less abundant mTECs in NIKaly/aly mice (10), TECs purified from NIKaly/aly thymus showed reduced expression of CAT-S (Table I): the ratio between the values from NIKaly/+ mice and NIKaly/aly mice was 0.12. When RNAs extracted from purified TECs were tested for TSA expression by real-time PCR using HPRT as an internal control, the difference between NIKaly/+ and NIKaly/aly mice became subtle when compared with the results obtained from total thymus, except for CRP (Table I). This finding is more obvious when taking into account that TECs purified from NIKaly/aly thymus contained reduced mTEC components compared with those from NIKaly/+ thymus, as revealed by the reduced expression of CAT-S. These results suggest that NIK regulates thymic expression of TSAs predominantly through the developmental process of mTECs, and not through transcriptional control of TSA genes within developed mTECs.


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  		Table I. Expression of tissue-specific genes in the thymusa

 
To further confirm this finding, we used mTEC lines established from the thymus. Because we were not able to establish TEC lines from IKK{alpha}–/– embryos at 18.5 days postcoitus for unknown reasons, we used mTEC lines established from NIKaly/aly embryos for this purpose; it is possible that the protein kinase-independent function of IKK{alpha} may contribute to the growth disadvantage of TECs lacking IKK{alpha} (23, 24). We established three cell lines from NIKaly/aly embryonic thymus (5-1, 4-1-3, and 6-5T), and these cells were positive for Ep-CAM, an epithelial cell marker, and staining with keratin-5 and ER-TR5 (Fig. 5A), but negative for keratin-8 and Th-3 (Ref.26 and Y. Mouri, M. Kasai, and M. Matsumoto, unpublished observation), consistent with a medullary origin. The TEC origin of these lines was also verified by Foxn1 expression (Fig. 5C). In these NIKaly/aly mTECs, agonistic stimulation of LTbetaR by mAb AF.H6 (29) did not induce NF-{kappa}B2 processing, in contrast to the mTECs derived from wild-type C57BL/6 mice (26) (Fig. 5B); LTbetaR ligation on wild-type mTEC increased amount of p52 in the nucleus (Nucl; bottom panel) with a concomitant reduction of p100 in the cytoplasm (Cyto; top panel), whereas the same treatment induced no nuclear p52 in NIKaly/aly mTEC. In contrast, CD40 ligation on both wild-type and NIKaly/aly mTECs had no such effect, which is consistent with the fact that CD40–/– mice showed undisturbed thymic architecture with normal distribution of mTECs containing UEA-1+ cells, ER-TR5+ cells, and Aire+ cells (Y. Mouri and M. Matsumoto, unpublished observation). Thus, these established cell lines show many of the characteristics of mTECs while retaining the features of aly-type NIK mutation.


Figure 5
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  		FIGURE 5. Retained expression of TSA genes in mTEC lines established from NIKaly/aly thymus. A, TEC lines established from NIKaly/aly embryos (5-1, 4-1-3, and 6-5T) were positive for Ep-CAM (top panels, stained in red), keratin-5 (middle panels, stained in green), and ER-TR5 (bottom panels, stained in red). Mouse embryonic fibroblasts (MEF) served as negative control. DNA staining is with 4'6-diamidino-2-phenylindole (stained in blue). Original magnification, x200. B, In wild-type mTEC (1C6), but not in NIKaly/aly mTEC (6-5T), LTbetaR ligation with agonistic anti-LTbetaR mAb increased amount of nuclear p52 (Nucl; bottom panel) with a concomitant reduction of p100 in the cytoplasm (Cyto; top panel). Such effect was observed in neither wild-type nor NIKaly/aly mTECs upon CD40 stimulation. One representative result from a total of three repeats is shown. Asterisks denote nonspecific bands. C, Semiquantitative RT-PCR for peripheral tissue-specific genes (SP1; FABP) was performed using mTECs established from control and NIKaly/aly thymus. The TEC origin of these cell lines was verified by Foxn1 expression. beta-actin was used to verify equal amounts of RNAs in each sample. One representative result from a total of three repeats is shown. Asterisks denote nonspecific bands. –, Without template.

 
mTEC lines established from NIKaly/aly mice showed levels of TSAs that were indistinguishable from those of control mTECs (Fig. 5C): SP1 and FABP were expressed from all the lines from both wild-type and NIKaly/aly mTECs. Expression of CRP assessed with real-time PCR was also indistinguishable between wild-type and NIKaly/aly mTECs: CRP/HPRT values from wild-type mTECs were 4.93 (line 2-3), 1.04 (line 1C6), and 2.21 (line 3-10), and CRP/HPRT values from NIKaly/aly mTECs were 2.75 (line 5-1), 1.67 (line 4-1-3), and 3.01 (line 6-5T). Insulin/HPRT values from wild-type mTECs were 0.47 (line 1C6) and 1.66 (line 3-10), and insulin/HPRT values from NIKaly/aly mTECs were 0.50 (line 5-1), 0.33 (line 4-1-3), and 0.65 (line 6-5T). Taken together, these results suggest that NIK is not required in individual mTECs for the transcriptional control of TSA genes. Rather, reduced expression of TSAs in NIKaly/aly thymus is most likely due to the developmental effect of mutated NIK on mTECs, leading to reduced absolute numbers of mTECs, each expressing normal levels of TSAs. Based on the similarity of autoimmune phenotypes between NIKaly/aly thymus and IKK{alpha}–/– thymus, we speculate that IKK{alpha} regulates TSA expression in the thymus through a developmental effect similar to that of NIK.


   Discussion
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We have demonstrated that IKK{alpha} plays an essential role in the organization of the thymic microenvironment that is required for the establishment of central tolerance. Grafting the thymic stroma from IKK{alpha}–/– mice onto athymic nude mice led to the development of autoimmune disease in the recipients; this also occurred in another group of recipient mice when the splenocytes from the IKK{alpha}–/– thymus-grafted mice were transferred. The thymic microenvironment that caused autoimmune disease in an IKK{alpha}-dependent manner was associated with structural abnormality (lack of UEA-1+ cells, and sparse ER-TR5+ and Aire+ cells in the medulla), defective NF-{kappa}B2 activation (impaired processing of p100 into p52), and reduced expression of TSAs. Because those phenotypes were similarly observed in NIKaly/aly mice (10), it is reasonable to speculate that the NIK-IKK{alpha} axis constitutes an essential step in this action, as demonstrated for secondary lymphoid organogenesis through LTbetaR involving NF-{kappa}B2 processing (7, 20, 37).

We have suggested that impaired processing of p100 into p52 caused by mutated NIK (10) or a lack of IKK{alpha}, as demonstrated in the present study, is relevant to the developmental defect of the thymic microenvironment. This reasoning is apparently inconsistent with the fact that mice deficient for p52 show no major defect in the thymus (38, 39). We interpret this discrepancy as a dominant effect of p100 on NF-{kappa}B activation in thymic stroma; accumulation of p100, rather than absence of p52, might be responsible for the thymic phenotypes we observed. In fact, mice lacking the COOH-terminal ankyrin domain of NF-{kappa}B2 (i.e., p100), but still containing a functional p52 protein, show abnormal development of the thymus (40), indicating the relevance of p100 to the control of thymic organogenesis. Notably, the mice deficient for p52 described above lack the whole NF-{kappa}B2 protein (including p100) because of the targeted deletion of the NF-{kappa}B2 gene locus (38, 39). We therefore consider that the ratio between p100 and p52 is a critical determinant for proper activation of the NF-{kappa}B complex that contains RelB as a heterodimeric partner (see below). Accumulation of p100 could disturb the nuclear localization of activated NF-{kappa}B complex within mTECs.

Although the exact mechanism by which IKK{alpha} regulates the thymic microenvironment that is required for the establishment of central tolerance is unknown, the existence of disorganized thymic structure together with an autoimmune disease phenotype in mice with a mutation disrupting the RelB gene merits attention. Because of the phenotypic similarities between NIK mutant mice and RelB–/– mice (41) (multi-inflammatory lesions together with the absence of UEA-1+ mTECs), together with the roles of IKK{alpha} demonstrated in the present study, we speculate that NIK-IKK{alpha} regulates the thymic microenvironment through activation of the NF-{kappa}B complex containing RelB. A requirement for NIK for activation of the NF-{kappa}B complex containing RelB is also seen in the production of NK T cells (41, 42). Interestingly, TNFR-associated factor 6 (TRAF6) in TECs is a critical component that regulates RelB expression, thereby controlling the thymic microenvironment for the establishment of central tolerance (43). Although both NIK-IKK{alpha}-dependent and TRAF6-dependent signals merge at the level of the NF-{kappa}B complex (i.e., p52/RelB), it is reasonable to speculate that the upstream receptors of each signal are distinct, because many NIK-IKK{alpha}-dependent signals are TRAF6 independent, as exemplified for LTbetaR (43), and vice versa. These results suggest the existence of a group of receptor-mediated signals that together control thymic organogenesis. The mechanisms that control the specificity of the heterodimeric complex of Rel family members (e.g., p52/RelB or p50/RelA) according to cell type and/or cellular signals also need to be clarified by future studies.

Signaling through LTbetaR has been demonstrated recently to control thymic organogenesis (44). However, because NIKaly/aly mice show more severe phenotypes of thymic structure than do LTbetaR-deficient mice (44), it would be reasonable to speculate that the NIK-IKK{alpha} axis is acting downstream of additional receptor(s) beyond LTbetaR in thymic organogenesis. Because CD40 is expressed on TECs (45, 46), and NF-{kappa}B2 processing takes place downstream of CD40, at least in B cells (34), CD40 could be a good candidate for an additional receptor that acts in NIK-IKK{alpha}-dependent thymic organogenesis. However, CD40–/– mice showed undisturbed thymic architecture with normal distribution of mTECs containing UEA-1+ cells, ER-TR5+ cells, and Aire+ cells (Y. Mouri and M. Matsumoto, unpublished observation), suggesting that CD40 alone is not responsible for this action. Consistent with this finding, CD40 ligation on wild-type mTEC (and NIKaly/aly mTEC as well) induced no NF-{kappa}B2 processing (Fig. 5B), although flow cytometric analysis clearly demonstrated CD40 expression on both mTEC lines (S. Niki and M. Matsumoto, unpublished observation). A complete description of the upstream receptor(s) required for thymic organogenesis in a NIK-IKK{alpha}-dependent manner is essential for a better understanding of the roles of NF-{kappa}B in the establishment of central tolerance.

The cellular mechanism controlling the establishment of self-tolerance in an IKK{alpha}-dependent manner is of considerable interest. Because of the perinatal death of IKK{alpha}–/– mice, we have investigated most of the IKK{alpha}-dependent autoimmune disease process with thymic chimeras. Because the autoimmune disease phenotype in NIKaly/aly mice is a result of both impaired elimination of autoreactive T cells and impaired production of Tregs (10), we suggest similar mechanisms for the breakdown of self-tolerance in the thymic microenvironment lacking IKK{alpha}. Consistent with this hypothesis, when control thymus and IKK{alpha}–/– thymus were grafted simultaneously onto BALB/cnu/nu mice, the development of inflammatory lesions was not completely inhibited (D. Kinoshita, K. Izumi, and M. Matsumoto, unpublished observation), suggesting that the grafted IKK{alpha}–/– thymus allows production of more pathogenic autoreactive T cells in the recipient mice than can be controlled by the Tregs that are produced by the grafted control thymus. We speculate that thymic stroma that has developed in the absence of IKK{alpha} may not be able to present TCR ligands (most likely containing self peptides) efficiently enough, resulting in insufficient avidity for the elimination of autoreactive T cells and/or production of Tregs (13, 14).

The autoimmunity that developed in NIKaly/aly mice (10) and IKK{alpha}–/– mice, described in the present study, was associated with altered expression of self Ags in the thymus, although the significance of this finding requires further study. We investigated whether reduced expression of self Ags in a NIK-IKK{alpha}-dependent manner was due to a reduction in the number of mTECs expressing these Ags or a lack of TSA gene transcription in these cells. Because purified TECs from NIKaly/aly thymus largely restored TSA expression, and the levels of TSAs expressed by mTEC lines isolated from NIKaly/aly mice were indistinguishable from the levels expressed by wild-type mTEC lines, the reduced TSA expression by total NIKaly/aly thymus is most likely due to the effect of the NIK-IKK{alpha} axis on the development of mTECs. Consistent with this finding, sorted TECs from LTbetaR-deficient mice (which have thymic disorganization and absolute reduction of TEC number) demonstrated unaltered expression of TSA genes (44). In contrast, Aire affects TSA expression without any obvious structural abnormalities of the thymus (16, 17). Thus, TSA expression in mTECs is controlled by a group of genes through their unique actions. Identification of particular cell types responsible for TSA expression, together with the nature of the TCR ligands (possibly TSA gene products) required for the establishment of self-tolerance, awaits further study. With the advent of thymic organogenesis using thymic precursor cells (47, 48), it may be feasible to manipulate the thymic microenvironment through the modulation of NF-{kappa}B activation pathways, thereby controlling the processes for the establishment of self-tolerance.


   Acknowledgments
 
We thank Drs. W. van Ewijk and M. Itoi for mAb ER-TR5, and Dr. P. D. Rennert for mAb AF.H6. We also thank Drs. H. Nakano and J. Inoue for valuable suggestions.


   Disclosures
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 This work was supported in part by Special Coordination Funds of the Ministry of Education, Culture, Sports, Science, and Technology, the Japanese Government, and by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, the Japanese Government (17047028 and 17390291). Back

2 Address correspondence and reprint requests to Dr. Mitsuru Matsumoto, Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail address: mitsuru{at}ier.tokushima-u.ac.jp Back

3 Abbreviations used in this paper: IKK, I{kappa}B kinase; Aire, autoimmune regulator; aly, alymphoplasia; CAT-S, cathepsin S; CRP, C-reactive protein; 2-DG, 2'-deoxyguanosine; FABP, fatty acid-binding protein; HPRT, hypoxanthine phosphoribosyltransferase; LT, lymphotoxin; mTEC, medullary thymic epithelial cell; NIK, NF-{kappa}B-inducing kinase; SP1, salivary protein 1; TEC, thymic epithelial cell; TRAF, TNFR-associated factor; Treg, immunoregulatory T cell; TSA, tissue-specific Ag; UEA, Ulex europaeus agglutinin; Ep-CAM, epithelial cell adhesion molecule; GAD67, glutamic acid decarboxylase 67. Back

Received for publication July 19, 2005. Accepted for publication January 26, 2006.


   References
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 Abstract
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 Discussion
 Disclosures
 References
 

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