No abstract

IntroductionB lymphocyte development in the bone marrow features a sequential rearrangement of the heavy and light chain loci and a transient expression of pre-B-cell receptor (pre-BCR). After a productive immunoglobulin heavy chain rearrangement at the pro-B stage, heavy chain protein mu pairs with the surrogate light chain (SLC) 5 and Vpre-B. Together with the signaling molecules Ig␣ and Ig␤, they form the pre-BCR on the cell surface. 1 The activation of the pre-BCR is cell autonomous and independent of ligand binding. 2 Signal emanated from the pre-BCR stimulates pre-B-cell proliferation and the formation of so-called large, cycling pre-B cells. After a limited number of cell divisions, cycling pre-B cells exit the cell cycle and become small, resting pre-B cells. Light chain rearrangement and transcription takes place primarily in those quiescent pre-B cells. Pre-BCR-induced B-cell self-propagation is an important event in B-cell development through which pre-B cells expressing successfully rearranged heavy chains are clonally expanded prior to light chain rearrangement. 3 In addition, pre-BCR signaling is also important for inhibiting the expression of Rag1 and Rag2, thus facilitating the maintenance of allelic exclusion of the heavy chain locus. 4 Moreover, pre-BCR signaling increases the accessibility of the light chain loci, thereby promoting light chain rearrangement and transcription. 5 The initial burst of cell proliferation at the large pre-B-cell stage and the subsequent passage into the quiescent, small pre-Bcell stage are critical events in pre-B-cell development. Disruption of the transition from large, cycling pre-B cells to small, resting pre-B cells often leads to a block in pre-B-cell development. [6][7][8] However, the molecular mechanisms that control pre-B-cell expansion, and therefore, the transition from cycling pre-B to resting pre-B cells, are still not clear. It has been shown that the pre-BCR is only expressed on cycling pre-B cells but not on small, resting pre-B cells. 9 Thus, down-regulation of pre-BCR has been linked to cessation of cell proliferation and cell-cycle withdrawal. 3,10 Ikaros and Aiolos are members of the Ikaros family of transcription factors. 11 The Ikaros family transcription factors interact with each other and other members of the Ikaros family. The N-terminal domain of Ikaros family proteins is responsible for DNA binding, whereas the C-terminal domain is involved in dimerization. The formation of Ikaros homo-and heterodimers through the C-terminal dimerization domain increases their affinity for DNA. 12,13 It has been demonstrated that expression of Ikaros and Aiolos are increased in pre-B cells relative to pro-B cells, suggesting that Ikaros and Aiolos may play an important role in pre-B-cell development. 14 Indeed, Aiolos has been shown to be directly involved in the silencing of the 5 gene in pre-B cells. 15 It has been reported that pre-BCR signaling induces the expression of Aiolos, which in turn, competes with EBF, an essential transcriptional activator...

Systemic lupus erythematosus (SLE) is a complex disease characterized by the appearance of autoantibodies against nuclear antigens and the involvement of multiple organ systems, including the kidneys. The precise immunological events that trigger the onset of clinical manifestations of SLE are not yet well understood. However, research using various mouse strains of spontaneous and inducible lupus in the last two decades has provided insights into the role of the immune system in the pathogenesis of this disease. According to our present understanding, the immunological defects resulting in the development of SLE can be categorized into two phases: (a) systemic autoimmunity resulting in increased serum antinuclear and antiglomerular autoantibodies and (b) immunological events that occur within the target organ and result in end organ damage. Aberrations in the innate as well as adaptive arms of the immune system both play an important role in the genesis and progression of lupus. Here, we will review the present understanding - as garnered from studying mouse models - about the roles of various immune cells in lupus pathogenesis.

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator in B cell development and function. We have previously shown that IRF4, together with IRF8, orchestrates pre-B cell development by limiting pre-B cell expansion and by promoting pre-B cell differentiation. Here, we report that IRF4 suppresses c-Myc induced leukemia in EμMyc mice. Our results show that c-Myc induced leukemia was greatly accelerated in the IRF4 heterozygous mice (IRF4+/−Myc); the average age of mortality in the IRF4+/−Myc mice was only 7 to 8 weeks but was 20 weeks in the control mice. Our results show that IRF4+/−Myc leukemic cells were derived from large pre-B cells and were hyperproliferative and resistant to apoptosis. Further analysis revealed that the majority of IRF4+/−Myc leukemic cells inactivated the wild-type IRF4 allele and contained defects in Arf-p53 tumor suppressor pathway. p27kip is part of the molecular circuitry that controls pre-B cell expansion. Our results show that expression of p27kip was lost in the IRF4+/−Myc leukemic cells and reconstitution of IRF4 expression in those cells induced p27kip and inhibited their expansion. Thus, IRF4 functions as a classical tumor suppressor to inhibit c-Myc induced B cell leukemia in EμMyc mice.

Objective Current treatment options for lupus are far from optimal. Previously, we reported that PI3K/AKT/mTOR, MEK1/Erk1,2, p38, STAT3, STAT5, NF-κB, multiple Bcl-2 family members, and various cell cycle molecules were over-expressed in splenic B-cells in an age-dependent and gene-dose-dependent manner in mouse strains with spontaneous lupus. As the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) has been shown to inhibit AKT, MEK1/2, and NF-κB, and to induce caspase-mediated apoptosis, we tested the therapeutic potential of this agent in murine lupus nephritis. Methods The synthetic triterpenoid CDDO-Me, or placebo, was administered to two month old B6.Sle1.Sle3 mice or MRL.lpr mice, which develop spontaneous lupus. All mice were phenotyped for disease. Results CDDO-Me-treated mice exhibited significantly reduced splenic cellularity, with decreased CD4+ T-cells and activated CD69+/CD4+ T-cells compared to the placebo-treated mice. These mice also exhibited significant reduction in serum autoantibody levels, including anti-dsDNA and anti-glomerular antibodies. Finally, CDDO-Me treatment attenuated renal disease in mice, as marked by reduced 24-hour proteinuria, blood urea nitrogen, and glomerulonephritis. At the mechanistic level, CDDO-Me treatment dampened MEK1/2, ERK, and STAT3 signaling within lymphocytes and oxidative stress. Importantly, the NF-E2-Related Factor 2 (Nrf2) pathway was activated after CDDO-Me treatment, indicating that CDDO-Me may be modulating renal damage in lupus via the inhibition of oxidative stress. Conclusion These findings underscore the importance of AKT/MEK1/2/NF-κB signaling in engendering murine lupus. Our studies reveal that the blockade of multiple signaling nodes and oxidative stress may effectively prevent and reverse the hematological, autoimmune and pathological manifestations of lupus.

B-cell development in the bone marrow is characterized by sequential rearrangement of immunoglobulin (Ig) heavy-and light-chain loci through a somatic DNA rearrangement event called the V(D)J rearrangement. Although the total randomness of V(D)J rearrangement is essential for the diversification of the B-cell-receptor (BCR) repertoire, it also unavoidably brings autoreactivity to the repertoire of newly generated immature B cells. Indeed, it has been estimated that 40 to 60% of newly synthesized B cells are autoreactive (29). Central tolerance is the mechanism through which developing B cells are rendered nonreactive to self. Central tolerance consists of receptor editing, anergy, and deletion (29). During receptor editing, autoreactive B cells undergo prolonged V(D)J rearrangement to replace the autoreactive heavy and/or light chain (9, 40). Anergy is a mechanism through which the autoreactive B cells are rendered inactive and, thus, unable to harm the host (10). Clonal deletion is the process through which the autoreactive B cells are depleted from the repertoire (12, 30). Recent studies have indicated that clonal deletion operates as a default pathway to get rid of autoreactive B cells that cannot be rescued by receptor editing (11,14).Receptor editing at the immature B-cell stage is induced by a self-reactive BCR, and it can also be induced by a BCR with an insufficient amount of tonic signaling (18). Receptor editing is a process through which self-reactive heavy or light chain is replaced with a product of secondary V(D)J rearrangement (29). Secondary rearrangement occurs mainly at the Ig and loci. The murine locus contains four functional J elements: J1, J2, J4, and J5. During receptor editing, the primary VJ rearrangement can be replaced by secondary rearrangement between V and a downstream J element. Secondary rearrangement can also occur between V and a recombination sequence (RS) located ϳ25 kb downstream of the C or between a site located in the J-C intron and the RS (7). The RS rearrangement leads to functional inactivation of the whole locus and the initiation of Ig rearrangement (41). Interferon regulatory factor 4 (IRF-4) and IRF-8 are immune system-specific transcription factors that have been shown to play critical roles in innate and adaptive immunity (39). Previous studies have demonstrated that IRF-4 and -8 function redundantly to control pre-B-cell development (21). B-cell development is blocked at the pre-B stage in mice lacking IRF-4 and -8; mutant pre-B cells are hyperproliferative and defective in light-chain rearrangement and transcription (21). Recently, we have shown that IRF-4 and -8 induce the expression of Ikaros and Aiolos to downregulate pre-BCR and inhibit pre-B-cell expansion (22). In addition, we and others have also demonstrated that IRF-4 and -8 induce chromatin modifications at the locus, thereby promoting locus activation in pre-B-cell development (20,23). Thus, the roles of IRF-4 and -8 in pre-B-cell development are twofold: one is to limit pre-B-cell expansion and the o...

B cell central tolerance is a process through which self-reactive B cells are removed from the B cell repertoire. Self-reactive B cells are generally removed by receptor editing in the bone marrow and by anergy induction in the periphery. Interferon regulatory factor 8 (IRF8) is a critical transcriptional regulator of immune system development and function. A recent study has shown that marginal zone B cells and B1 B cells population are dramatically increased in the IRF8 deficient mice, indicating that there are B cell developmental defects in the absence of IRF8. Here, we report that mice deficient for IRF8 produced anti-dsDNA antibodies. Using hen egg lysozyme double transgenic model, we further demonstrate that B cell anergy was breached in the IRF8 deficient mice. While anergic B cells in the IRF8 proficient background were blocked at the transitional stage of development, anergic B cells in the IRF8 deficient background were able to further mature which allow them to regain responses to antigen stimulation. Interestingly, our results show that IRF8 deficient B cells were more sensitive to antigen stimulation and were resistant to antigen induced cell death. Moreover, our results show that IRF8 was expressed at a high level in the anergic B cells and elevated level of IRF8 promoted apoptosis in the transitional B cells. Thus, our findings presented here reveal a previously unrecognized function of IRF8 in B cell anergy induction.

SummarySystemic lupus erythematosus (SLE) is a polygenic autoimmune disease characterized by the production of anti-nuclear autoantibodies that lead to subsequent end organ damage. Previous array-based studies in patients with SLE have shown that high immunoglobulin (Ig)G anti-nuclear autoantibody reactivity was associated with severe renal lupus, whereas IgM polyreactivity was associated with less severe disease. To ascertain how different murine lupus strains recapitulate these different autoantibody profiles seen in patients, serum from New Zealand black (NZB)/NZ white (W) F1, Murphy Roths large (MRL)/lpr, NZ mixed (M)2410 and BXSB strains were compared using a comprehensive array-based screen. The array results were verified using enzyme-linked immunosorbent assays (ELISAs). Serum from MRL/lpr mice exhibited high levels of IgG anti-nuclear antibodies as well as anti-glomerular antibodies and variable levels of antibodies to myosin, Matrigel and thyroglobulin. Elevated anti-nuclear IgG antibodies were associated with severe nephritis in this strain. In contrast, NZM2410 mice exhibited lower IgG autoantibody levels with less severe nephritis but a significantly higher polyreactive IgM autoantibody profile. ELISA analysis confirmed these results. The NZB/NZW F1 and BXSB strains exhibited an intermediate serological profile. Hence, just as in patients with SLE, whereas strong IgG reactivity to nuclear antigens is associated with severe renal disease, a polyreactive IgM seroprofile is also less ominous in murine lupus.