NF-kB binding and mechanism of action. Thus, IkBa tends to bind p65/p50, but IkBs -e and -b prefer c-Rel and p65 [8,10,11,12,13,14]. Further, although IkBe associates with p50 and p52 in vivo and in vitro, it is DNA-binding of cRel/p50 and cRel or p65 dimers which it inhibits [15], suggesting that IkBe regulates genes transactivated by heterodimers of c-Rel and homodimers of RelA. Functionally, IkBe and IkBa both contribute to post-induction repression of transcription by removing NF-kB from the nucleus, whereas newly-synthesised IkBb forms a ternary complex with NF-kB on DNA to maintain its transcriptional activity [16,17]. Recent studies suggest that differential regulation of IkBe may also be effected through interactions with the protein phosphatase 6 (PP6) ternary complex [18,19,20]. c-Rel, the prototype member of the NF-kB family, is expressed in lymphoid, myelomonocytic and erythroid cells in haematopoi- etic organs of both adult and foetus, and is essential for normal function of B and T cells, macrophages and dendritic cells. Its expression and activation are regulated according to cell lineage, developmental and differentiation state, as well as stimulus received by the cell [21,22]. For example, c-Rel is constitutively cytosolic in na¨ıve B lymphocytes but nuclear in mature B cells, an altered state attributed to an increased rate of degradation of IkBa in mature B cells [23] but which perhaps better reflects the down- regulation of IkBe in the latter [9]. As a second example, the cytosolic pool of c-Rel in resting T lymphocytes is not translocated instantly to the nucleus following stimulation via the T cell receptor (TCR). Rather, newly synthesised c-Rel only appears in the nucleus after 1.522 hours of TCR activation [24]. This study, which predated the discovery of IkBe, suggested that differential subunit- and cell-specific regulation of NF-kB was effected by IkBa and -b. Thus, mechanisms of temporal and spatial regulation by IkB of c-Rel have not been fully defined in lymphocytes. c-Rel upregulates genes involved in survival, proliferation or differentiation including c-Rel itself, interleukin- 2 (IL-2) in T cells, and genes involved in B cell survival [22,25,26]. AP-1 and NFAT transcription factors are also required for full activation of the IL-2 proximal promoter (pIL-2), but c-Rel is essential for IL-2 expression [27,28,29,30]. c-Rel is required for induction of CD40. CD40 was originally detected on B lymphocytes but has since been shown to be expressed on T cells and antigen presenting cells such as dendritic cells, as well as non-haemato- poietic endothelial cells and fibroblasts [26,31,32,33]. Ligation of CD40 on B cells delivers anti-apoptotic, maturation and proliferative signals to the B cell [34]. Prolonged activation of c- Rel, such as occurs during B cell maturation, also promotes B cell receptor-dependent induction of B cell activating factor receptor (BAFF-R) and its downstream target p100/NF-kB2, rendering B cells competent for the anti-apoptotic BAFF signal [35,36]. Furthermore, both CD40 and BAFF-R have been reported to interact and cooperate with nuclear c-Rel, but not p65, to upregulate their own ligands on B cells [37,38]. An earlier focus of our laboratory has been the study of the effects of tumour necrosis factor-a (TNF), whose expression is dysregulated in inflammatory diseases such as rheumatoid arthritis, on T cell function [39,40]. Specifically, we wanted to elucidate mechanisms whereby TNF caused profound, reversible inhibition of subsequent IL-2 induction in mouse T cell hybridomas [41,42]. In the work presented here, we show that induction of IL-2 was suppressed at the level of transcription and that TNF inhibited PMA and ionomycin-induced expression, nuclear translocation and transcriptional activity of AP-1, NFAT and NF-kB to a greater or lesser extent. TNF pre-treatment also inhibited NF-kB signalling when cells were subsequently restim- Regulation of c-Rel by IkBe in Murine Lymphocytes ulated with TNF. Thus prolonged culture in TNF affected 11A2 T cell signalling in a number of ways, many of which were likely to contribute to the profound suppression of IL-2 production In the course of these studies, however, we became most interested in the two clearest and most consistent effects of TNF, which were a very marked inhibition of nuclear translocation of c- Rel, compared to p65, AP-1 or NFAT and, secondly, selective upregulation and impaired degradation of IkBe, out of the IkBs. We wondered to what extent the first phenomenon was a consequence of the second and wanted to explore whether this was an example of selective regulation of c-Rel by IkBe in lymphocytes. Our hypothesis was that, in the absence of IkBe, c- Rel would appear in the nucleus of unstimulated and na¨ıve lymphocytes and the cells would therefore show altered expression of c-Rel-dependent genes. We found increased nuclear expression of c-Rel in unstimulated T cell blasts and in na¨ıve splenic B cells from IkBe-deficient mice, despite increased basal expression of IkBa in these mice. IkBe deficiency was associated with increased lymph node cellularity and basal thymidine incorporation of lymphoid cells ex vivo. We observed increased lymph node B cell numbers in vivo and enhanced B lymphocyte survival ex vivo which was associated with upregulation of CD40 and BAFF-R in IkBe2/2 mice. The data suggest that negative regulation of these c-Rel-dependent pro-survival genes is a non-redundant function of IkBe in B cells. We propose that modulation of IkBe expression and degradation is an important mechanism whereby the fine- tuning of appropriate c-Rel activity is achieved in lymphoid cells. Materials and Methods Antibodies and reagents Agonistic anti-CD3e mAb 145-2C11, and rat anti-mouse IL-2 antibody pairs were from BD-Biosciences, Oxford, UK. Antibod- ies against c-Fos, Fos B, Fra-1, Fra-2, c-Jun, Jun B, Jun D, NFAT2 (NFATc1, clone 7A2), p65/RelA, c-Rel, p50 (NLS), and IkBs -a, -b and -e, used for immunoblots, EMSA supershift and immunoprecipitation, were all from Santa Cruz Biotechnology (Insight Biotechnology, Wembley, UK). Agarose-conjugated anti- c-Rel and anti-p65 Abs for immunoprecipitation were also from Santa Cruz. Anti-NFAT1 (NFATp, NFATc2) antibody was from Affinity BioreagentsTM (Thermo Fisher Scientific, Loughborough, UK). HRP-linked antibodies for immunoblot were all from DAKO (DAKO UK, Ely, UK). All flow cytometry antibodies were from eBioscience (eBioscience Ltd, Hatfield, UK). EMSA oligonucleotide probes: AP-1 59-CGCTTGATGAGTCAGCCG- GAA-39; NF-kB 59-AGTTGAGGGGACTTTCCCAGG-39 were both from Promega (Promega UK, Southampton, UK); CD28RR 59-TTTAAAGAAATTCCAAAGAGTCATCA-39 (forward and reverse primers from MWG (Eurofins MWG Operon, Ebersberg, Germany); NFAT/AP-1 59-CGCCCAAAGAGGAAAATTTG- TTTCATA-39 (Santa-Cruz/Insight Biotechnology, Wembley, UK). Other reagents were from Sigma-Aldrich Company Ltd (Dorset, UK) or BDH (VWR International, Lutterworth, UK). Cells and Cell Culture The derivation and culture of mouse T cell hybridoma clone 11A2, which is specific for human cartilage glycoprotein-39 (HCgp-39), restricted by HLA-DR4, and expresses human CD4, has been described [43]. 11A2 cells were cultured in RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 10% heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate and 50 mM 2-mercapto- ethanol, at 37uC and 5% CO2. Cells were passaged every 48 hours into fresh complete medium, in the presence or absence PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24504