IL-7 does not prevent pro-B/pre-B cell maturation to the immature/sIgM(+) stage

IL-7 plays many fundamental roles during murine B lineage development. One reported function is to maintain progenitors in a developmentally immature state by preventing differentiation to the surface IgM (sIgM)(+) stage. Withdrawal of IL-7 from cultures has been shown to lead to increases in mature traits such as RAG expression, IgL rearrangements and expression of sIgM. These observations have been interpreted as an inductive event promoting the differentiation of progenitor cells. In contrast to this, we reproducibly observe sIgM(+) cells that have differentiated in cultures containing IL-7. We find that sIgM(+) cells arise as a normal consequence when B lineage cells are cultured in the presence of IL-7. However, these cells are short-lived and are quickly replaced by newly emerging sIgM(+) cells that differentiate from sIgM(-) progenitors. Withdrawal of IL-7 from these cultures only prevents the survival and proliferation of CD2(-)sIgM(-) pro-B cells but does not change the number of cells that differentiate to the sIgM(+) stage. This changes the ratio of sIgM(-):sIgM(+) cells and results only in an apparent maturation of the culture as a whole. Therefore withdrawal of IL-7 from these cultures acts as a selection event, not an induction event, for populations that are normally present.     

IL-7: a key regulator of B lymphopoiesis

Interleukin-7 plays important roles in the B cell developmental pathway including events leading to commitment, survival, proliferation, and maturation. Because of its central role in adult murine B lymphopoiesis, IL-7 is frequently used to generate B cell progenitors in vitro. We have shown that differentiation of IL-7-responsive cells in these cultures is influenced by CD45, pre-B cell receptor, and other downstream signaling molecules. A common, but often overlooked aspect of IL-7 containing cultures is the routine maturation of cells to the sIgM(+) stage. The production of B cells in IL-7 containing cultures is balanced by cell death, since such cells fail to survive for long without additional stimulation.     

Gene transfer of the interleukin (IL)-2 receptor β chain into an IL-7-dependent pre-B cell line permits IL-2-driven proliferation: Tyrosine phosphorylation of Shc is induced by IL-2 but not IL-7

Expression of the interleukin (IL)-2 receptor β chain in the IL-7-dependent pre-B cell line I × N/2B permitted growth in presence of either IL-2 or IL-7, allowing for a direct comparison of intracellular signaling events. Protein tyrosine phosphorylation was essential for IL-2- and IL-7-induced signal transduction since the tyrosine kinase inhibitor herbimycin A blocked proliferation in response to both factors. Western blot analysis of tyrosine-phosphorylated proteins revealed that both IL-2 and IL-7 stimulation led to enhanced phosphorylation of proteins of 170-, 145, 115- and 99-kDa, as well as induction of phosphorylation of a 96-kDa protein. However, a 55- and a 155-kDa protein were only phosphorylated after IL-2 stimulation. The 55-kDa protein specifically phosphorylated by IL-2 could be identified as p52^shc which has recently been shown to be critically involved in Ras activation. Shc tyrosine phosphorylation as a result of IL-2 stimulation was consistently found in CTLL-2 cells and human T lymphoblasts. Taken together our results indicate that the IL-2- and IL-7-stimulated intracellular pathways are partially different and that Shc is a target of IL2-, but not IL-7-, stimulated tyrosine phosphorylation.     

Bruton's tyrosine kinase is required for signaling the CD79b-mediated pro-B to pre-B cell transition

Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.     

IL-4 and IL-2 upregulate the expression of antigen B7, the B cell counterstructure to T cell CD28: an amplification mechanism for T-B cell interactions.

We recently generated mAb 104 which is specific for the B cell activation antigen Ag B7. With this we studied the regulation of Ag B7 expression on normal tonsillar B lymphocytes as well as the activities of B7+ and B7- activated B cells. SAC and to a lesser extent anti-IgM antibody upregulated Ag B7 and this was further enhanced by IL-2 and most notably IL-4. Ag B7 was expressed on virtually all sIgG+ and sIgA+ B cells and approximately half of the sIgD+ and sIgM+ B cells. SAC-stimulated B7+ B cells proliferated and produced IgM, IgG and IgA in response to IL-2 and IgM and IgG in response to IL-4. SAC-stimulated B7- B cells proliferated and produced only IgM in response to IL-2 and IL-4. Considering that Ag B7 has recently been shown to be the counterstructure of the T cell CD28 and that CD28 triggering strongly enhances cytokine production by T cells, it is likely that the CD28/B7 interaction represents an important amplification phenomenon in T-B cell interaction leading to humoral immune responses. The preferential expression of Ag B7 on IgG and IgA committed cells suggests that CD28/B7 interaction may be more specific to secondary antibody responses provided by memory T and B cells.     

Interleukin-7 specifically induces the B7/BB1 antigen on human cord blood and peripheral blood T cells and T cell clones

B7/BB1 is a cell surface molecule and member of the Ig superfamily that is constitutively expressed on dendritic cells.In addition, B7 is expressed on B cells, macrophages, T cells,and T cell clones following activation. Interaction of B7 withits natural ligand CD28 is required for optimal stimulationof T cells, activated via the TCR-CD3 complex, which is thoughtto be due to stabilization of cytokine mRNA. Here we demonstratethat the expression of B7 on T cells can specifically be inducedby IL-7. Induction of B7 expression on T cells and T cell clonesrequires at least 5 – 7 days of culture and representsa late activation event. Results of studies using T cell clones,as well as resting purified B7^– T cells, demonstrate thatB7 is induced on a substantial proportion of T cells after IL-7activation and is not due to an outgrowth of pre-existing B7+T cells. In addition, CD4+ as well as CD8+ T cells could beinduced to express B7. Stimulation of purified cord blood Tcells with cross-linked anti-CD3 mAb resulted in a relativelyfast (48 h) induction of B7, which could not be inhibited bya neutralizing anti-IL-7 mAb, whereas no endogenous IL-7 productionby activated T cells and T cell clones could be detected. Together,these results indicate that the B7 molecule can be induced onT cells by IL-7, but also by an IL-7 independent pathway involvingtriggering of the TCR-CD3 complex.     

Keeping track of pro-B cells: a new model for the effects of IL-7 during B cell development

Trophic and instructive models have been invoked to explain how various cytokines influence the survival, proliferation and differentiation of lymphoid progenitors. There is evidence that IL-7 can act in either a trophic or an instructive manner during B and T cell development although its roles in B cell development in the fetus versus the adult mouse and in the mouse versus the human may vary. Here we outline the stages of B cell development and the conventional model of the role of IL-7 in the transitions from pro-B cells to pre-B cells and then to immature B cells. We also discuss the implications of recent data that have led to a new model for this process.     

IGFBP7对人乳腺癌MCF-7细胞增殖的影响及其机制

目的: 探究胰岛素样生长因子结合蛋白7(IGFBP7)对人乳腺癌MCF-7细胞增殖的影响及其分子生物学机制。方法: 将质粒pCMV6-IGFBP7转染MCF-7细胞,构建稳定表达IGFBP7的MCF-7细胞系;采用Western blotting检测IGFBP7在MCF-7细胞稳定转染子的表达;采用软琼脂培养克隆形成实验检测IGFBP7对MCF-7细胞克隆形成能力的影响;采用流式细胞术检测IGFBP7对MCF-7细胞周期的影响;采用Western blotting检测IGFBP7对MCF-7细胞细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、细胞周期素D1(cyclin D1)、细胞周期素依赖性激酶4(CDK4)、cyclin E、CDK2、p21^CIP1/WAF1、p27^KIP1、p53、视网膜母细胞瘤蛋白(Rb)和p-Rb蛋白含量的影响。结果: (1)只有稳定转染质粒pCMV6-IGFBP7的MCF-7细胞表达IGFBP7。(2)IGFBP7能够显著降低MCF-7细胞的克隆形成率(P<0.01),阻止细胞从G₁期进入S 期,使其停滞于G₁期(P<0.01)。(3)IGFBP7能够显著抑制ERK1/2的磷酸化(P<0.01)。(4)IGFBP7能够下调cyclin D1和cyclin E蛋白表达(P<0.01),上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达(P<0.01),抑制Rb的磷酸化(P<0.01)。(5)MEK1/2阻断剂PD98059可部分模拟IGFBP7的肿瘤抑制效应。结论: (1) IGFBP7可通过下调cyclin D1和cyclin E蛋白表达,上调p27^KIP1、p21^CIP1/WAF1和p53蛋白表达,以及抑制Rb磷酸化发挥抗肿瘤作用;(2) IGFBP7对cyclin D1和p27^KIP1的调节可能与其抑制ERK1/2信号通路有关。     

PAX5 promotes pre-B cell proliferation by regulating the expression of pre-B cell receptor and its downstream signaling

PAX5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. Although previous studies have indicated that the Pax5-conditional-knockout mouse exhibited dedifferentiation of mature B cell and the development of aggressive lymphomas, the changes of Pax5 gene expressions in pre-B cells have not been analyzed. To understand the functional importance of Pax5 gene in the proliferation and survival of pre-B cells, we established a Pax5-knockdown model using 70Z/3 pre-B cell line. Pax5 knockdown 70Z/3 cells (70Z/3-KD cells) showed down-regulations of pre-BCR compounds such as CD19, BLNK, Id2 and λ5. The signaling via pre-BCRs was significantly diminished in the 70Z/3-KD cells, and this alteration was normalized by restored Pax5 gene expression. Loss of PAX5 reduced the growth rates in the 70Z/3-KD cells, compared to the mock cells. Meanwhile, the proliferation of pre-B cells was reduced by the knockdown of Pax5 gene. Moreover, further examinations showed that PAX5 was also activated in B cell acute lymphoblastic leukemia (B-ALL) as a cell proliferation enhancer. These findings suggested that pax5 is critically important for the proliferation and survival of pre-B cells.     

The skewed heavy-chain repertoire in peritoneal B-1 cells is predetermined by the selection via pre-B cell receptor during B cell ontogeny in the fetal liver

As many as 5–15% of B-1 cells in the peritoneal cavityof adult mice produce antibodies reactive to phosphatidylcholine(PtC) and the vast majority of them express B cell receptors(BCRs) composed of V_H11-µH chains utilizing the J_H1 segmentand V9-L chains. This extremely skewed repertoire of PtC-reactiveB-1 cells is traditionally attributed to the expansion of particularclones in response to self or exogenous antigens. Here, we showthat the strong bias toward the J_H1 usage among V_H11-µHchains is already established prior to the BCR assembly, namelyat the transition from the large to the small pre-B cell stageduring B cell ontogeny in the fetal liver. Among V_H11-µHclones isolated from large pre-B cells where the J_H1 skewingwas not established yet, the J_H1 users showed the highest abilityto form pre-B cell receptor (pre-BCR) and to induce cellularproliferation and differentiation when expressed in fetal liverpro-B cells. Thus, the J_H1 users were positively selected andamplified at the pre-BCR checkpoint. When co-expressed withV9-L chains to form BCR, the J_H1 users almost exclusively conferredthe PtC reactivity on BCR even though other J_H users could alsoform BCR on the cell surface. Therefore, the pre-BCR-mediatedpositive selection of the J_H1 users among V_H11-µH chainsappears to be beneficial to the efficient generation of ‘innate-type’PtC-reactive B cells during the fetal B cell development, evenbefore the self-renewal or the antigen-driven clonal expansionof B-1 cells takes place in the peritoneal cavity.     

Pre-B cell receptor signaling mediates selective response to IL-7 at the pro-B to pre-B cell transition via an ERK/MAP kinase-dependent pathway.

B lymphocyte development is regulated at multiple checkpoints, mediated by signals originating both inside and outside the cell. Two signaling pathways known to be essential in this process are interleukin-7 (IL-7) and the pre-B cell receptor (pBCR). We have shown previously that these signaling pathways intersect functionally. Specifically, response to low concentrations of IL-7 requires pBCR expression. In this report, we identify the ERK/MAP kinase pathway as a key regulatory component of this response. We propose a molecular mechanism for the selective expansion of pBCR(+) precursors and for the culling of inappropriately rearranged pro-B cells.     

Development of a continuous IL-7-dependent murine pre-B cell line PB-1 suitable for the biological characterisation and assay of human IL-7

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness. The cells survive freezing and reviving, having been stored for periods of up to 4 years. The IL-7 bioassay is reproducible and sensitive, able to reliably detect 50 pg/ml IL-7. The assay is completely unresponsive to any other stimulatory cytokines tested and is not affected by a wide variety of inhibitory cytokines, with the exception of high levels of interferon alpha. The assay can be made completely specific for human IL-7 by including specific neutralizing antibodies for IL-7 and has been shown to be suitable for the estimation of IL-7 in both plasma and serum samples.     

乙肝疫苗无、弱应答与B7-CD28及IL-12、IL-10的相关性

目的：研究乙肝疫苗无、弱应答与B7-CD28分子及IL-12、IL-10的相关性。方法：分离并培养乙肝疫苗强应答和无、弱应答者PBMCs，以PHA或rHBsAg刺激。用流式细胞术检测培养细胞CD80、CD86及CD28的表达；酶标法检测上清IL-12、IL-10的水平；MTT法检测细胞增殖反应。结果：rHRsAg刺激后，无、弱应答组和强应答组比较，CD80和CD86的表达率、IL-12和IL-10水平、细胞增殖反应均降低，差异显著；但CD4^ 、CD8^ 细胞CD28的表达率无显著差异。PHA刺激后，上述指标在2组之间均无显著差异。结论：乙肝疫苗无、弱应答者一般的细胞免疫功能正常。抗-HBs低下与CD80、CD86表达及IL-12、IL-10产生不足等有关，但无T细胞CD28的表达缺陷。     

Subcellular receptor redistribution and enhanced microspike formation by a Ret receptor preferentially recruiting Dok

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret^Dok+) with little or no remaining interactions with Shc and Frs2. Ret^Dok+ displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret^WT. We show that early events after ligand stimulation of Ret^Dok+ include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret^Dok+ receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.     

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77 %) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.     

Impaired B-1 and B-2 B cell development and atypical splenic B cell structures in IL-7 receptor-deficient mice

The cytokine IL-7 and its receptor are essential for normal B and T lymphopoiesis. We have analyzed the role of this receptor in B cell development throughout ontogeny in IL-7 receptor alpha-deficient mice. We demonstrate that the IL-7 receptor becomes progressively more important with age. B lymphopoiesis takes place, albeit at reduced levels, in fetal liver and bone marrow of young mice, but is arrested in adults. The outcome is a severe reduction, from an early age, in peripheral B cells including follicular, marginal zone and B-1 B cells as well as perturbed splenic B cell structures, which are restored after adoptive transfer of normal spleen cells. We conclude that in the absence of the IL-7 receptor, the residual B lymphopoiesis occurring early in ontogeny must be facilitated by another component, whereas the IL-7 receptor is the key factor in adults. The impairment of marginal zone and B-1 B cells in IL-7 receptor- but not IL-7-deficient mice suggests non-redundant functions for the IL-7 receptor ligands, IL-7 and thymic stromal lymphopoietin.     

IL-2 receptor {alpha} chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor(CD25.TAC) on the surface of B lineage cells In mouse bone marrowreveals that it is a useful marker to distinguish pre-B-I frompre-B-II cells. CD25 Is not expressed on CD45R(B220)⁺ c-kit⁺CD43⁺ TdT⁺ ₅⁺ C_µ^– slg^– lgH chain locus DJ_H-rearrangedpre-B-I cells of mouse bone marrow. It is expressed on largecycling CD45R(B220)⁺ c-kit⁺ CD43⁺ TdT⁺ ₅⁺ C_µ^– sig^–and on small resting CD45R(B220)⁺ c-kit⁺ CD43^– TdT⁺ ₅⁺C_µ^– sig^– sig- IgH chain locus VHDJH-rearrangedpre-B-II cells. Therefore, the transition from pre-B-I to largepre-B-II cells is marked by the downregulation of c-kit andterminal deoxynucleotldyl transferase (TdT), and by the upregulattonof CD25. SCID, RAG-2T, _µMT and ₆T mutant mice do havenormal, If not elevated numbers of pre-B-I cells but lack allCD25⁺ pre-B-II cells in their bone marrow. The expression ofa transgenic H chain under control of the _µH chain enhancerin RAG-2T bone marrow B lineage precursors allows the developmentof large and small CD25⁺ pre-B-II cells. The results suggestthat the differentiation of pre-B-I to pre-B-II cells in mousebone marrow requires the expression of _µH chains and surrogateL chains in membranes, probably on the surface of precursorB cells.     

SA4503, a sigma-1 receptor agonist,prevents cultured cortical neurons from oxidative stress-induced cell death via suppression of MAPK pathway activation and glutamate receptor expression

Many studies suggest that antidepressants act as neuroprotective agents in the central nervous system (CNS), though the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of SA4503, which is a sigma-1 receptor agonist and a novel antidepressant candidate, on oxidative stress-induced cell death in cultured cortical neurons. Exposure of the neurons to H₂O₂ induced cell death, while pretreatment with SA4503 inhibited neuronal cell death. The SA4503-dependent survival effect was reversed by co-application with BD1047 (an antagonist of sigma-1/2 receptors). Previously we found that H₂O₂ triggers a series of events including over-activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and intracellular Ca²⁺ accumulation via voltage-gated Ca²⁺ channels and ionotropic glutamate receptors, resulting in neuronal cell death (Numakawa et al. (2007) [20]). Importantly, we found in this study that SA4503 reduced the activation of the MAPK/ERK pathway and down-regulated the ionotropic glutamate receptor, GluR1. Taking these findings together, it is possible that SA4503 blocks neuronal cell death via repressing activation of the MAPK/ERK pathway and, consequently, expression levels of glutamate receptors.     

Persistent human immunodeficiency virus-1 antigenaemia affects the expression of interleukin-7Ralpha on central and effector memory CD4+ and CD8+ T cell subsets

Interleukin (IL)-7 and its receptor (IL-7Ralpha) play important roles in regulating lymphopoiesis. Previous studies have reported that human immunodeficiency virus-1 (HIV-1) viraemia affects the expression of IL-7Ralpha, but its effects on CD4+ and CD8+ T cell memory subsets have not been studied. Using eight-colour flow cytometry, we compared the immunophenotypic patterns of CD4+ and CD8+ T cell subsets expressing IL-7Ralpha and activation markers, as well as circulating IL-7 levels, in three well-defined groups of HIV-1-infected subjects: successfully treated, viraemic and long-term non-progressor (LTNP). Compared with successfully treated and LTNP subjects, viraemic patients had reduced expression of IL-7Ralpha on both CD4+ and CD8+ T cells, particularly on central and effector memory T cell compartments, and substantially elevated expression of activation markers on CD8+ T cell subsets. Circulating IL-7 levels were correlated negatively with the number of CD4+ and CD8+ T cell subsets expressing IL-7Ralpha; these associations were stronger with CD4+ T cell subsets and mainly with central and effector memory cells. The expression of activation markers on CD4+ and CD8+ cell T subsets was not related to circulating IL-7 levels. A strong negative correlation was observed between central memory CD4+ or CD8+ T cells expressing IL-7Ralpha and those expressing activation markers, independently of IL-7 levels. Collectively, these results provide further insight on the role of unsuppressed viral load in disrupting the IL-7/IL-7Ralpha system and contributing to HIV-1 disease progression.