Senescent B lymphopoiesis is balanced in suppressive homeostasis: decrease in interleukin-7 and transforming growth factor-beta levels in stromal cells of senescence-accelerated mice

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-beta (TGF-beta) was found to contribute to B cell suppression.To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-beta during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7-responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-beta is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-beta antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 microg/ml). Furthermore, TGF-beta protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-beta concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-beta activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosuppression).     

Inflammatory biomarker, neopterin, suppresses B lymphopoiesis for possible facilitation of granulocyte responses, which is severely altered in age-related stromal-cell-impaired mice, SCI/SAM

Neopterin is produced by monocytes and is a useful biomarker of inflammatory activation. We found that neopterin enhanced in vivo and in vitro granulopoiesis triggered by the stromal-cell production of cytokines in mice. The effects of neopterin on B lymphopoiesis during the enhancement of granulopoiesis were determined using the mouse model of senescent stromal-cell impairment (SCI), a subline of senescence-accelerated mice (SAM). In non-SCI mice (a less senescent stage of SCI mice), treatment with neopterin decreased the number of colonies, on a semisolid medium, of colony-forming units of pre-B-cell progenitors (CFU-preB) from unfractionated bone marrow (BM) cells, but not that from a population rich in pro-B and pre-B cells without stromal cells. Neopterin upregulated the expression of genes for the negative regulators of B lymphopoiesis such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in cultured stromal cells, implying that neopterin suppressed the CFU-preB colony formation by inducing negative regulators from stromal cells. The intraperitoneal injection of neopterin into non-SCI mice resulted in a marked decrease in the number of femoral CFU-preB within 1 day, along with increases in TNF-alpha and IL-6 expression levels. However, in SCI mice, in vivo and in vitro responses to B lymphopoiesis and the upregulation of cytokines after neopterin treatment were less marked than those in non-SCI mice. These results suggest that neopterin predominantly suppressed lymphopoiesis by inducing the production of negative regulators of B lymphopoiesis by stromal cells, resulting in the selective suppression of in vivo B lymphopoiesis. These results also suggest that neopterin facilitated granulopoiesis in BM by suppressing B lymphopoiesis, thereby contributing to the potentiation of the inflammatory process; interestingly, such neopterin function became impaired during senescence because of attenuated stromal-cell function, resulting in the downmodulation of the host-defense mechanism in the aged.     

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.     

Effects of aging on the common lymphoid progenitor to pro-B cell transition

The number of common lymphoid progenitors (CLP) and their pre-pro-B and pro-B cell progeny is reduced in old mice, but the age-related changes responsible for these declines have not been fully elucidated. The aim of this study was to provide additional insights into the impact of senescence on early B cell development by analyzing the CLP and pro-B cell compartments under steady-state conditions and after cytoablation with 5-fluorouracil. 5-Fluorouracil subjects the hemopoietic system to acute stress and has the advantage of revealing defects in progenitors that may otherwise be subtle. The data demonstrate significant, age-related defects in the proliferative potential of early B cell precursors and suggest that the ability of CLP to differentiate into pre-pro-B cells is also compromised by senescence. These age-related changes in early B lymphopoiesis do not result from a general defect in HSC or the bone marrow microenvironment that impairs development in all hemopoietic lineages. Instead, data demonstrating that myeloid progenitor number and developmental potential do not decline with age indicate that B lymphopoiesis is particularly sensitive to defects that accumulate during senescence.     

Proteasome inhibition drastically but reversibly impairs murine lymphocyte development

The proteasome inhibitor bortezomib, which induces cell death in various cancer cell lines including lymphatic neoplasias, has recently been approved for the treatment of relapsed multiple myeloma. Important mechanisms of proteasome inhibitor-mediated tumor cell death are the inhibition of NF-kappaB activation and induction of the terminal unfolded protein response (UPR). However, little is known about effects of bortezomib on developing and mature lymphocytes. Therefore, Balb/C mice were injected with bortezomib and lymphocyte subsets were analyzed. This treatment resulted in dramatically decreased numbers of T and B lymphocyte precursors, while mature lymphocytes were only partially affected. Thymocytes were almost depleted 3 days after a single bortezomib injection, pro-B and pre-B cells already after 2 days. Thymocytes and B cell precursors recovered within 2 weeks. The decreased numbers of developing lymphocytes were due to apoptotic cell death accompanied by strongly increased caspase 3/7 activity. Within 8 h after bortezomib injection, there was a strong induction of heat shock protein 70 and C/EBP homologous protein in bone marrow B cells, indicating endoplasmic reticulum stress and activation of the terminal UPR, respectively. Hence, induction of apoptosis by proteasome inhibition can dramatically affect lymphocyte development, a fact which has important implications for the clinical use of bortezomib, especially in situations with ongoing lymphopoiesis.     

Comparative analysis of nuclear proteins of B cells in different developmental stages

The developmental stage-specific regulation of V(D)J recombination, a gene rearrangement process of antigen receptor gene segments, is tightly controlled in cells. Here we screened proteins uniquely or differentially expressed among three developmentally distinguishable B cells (pro-B, pre-B and mature B cells) using two-dimensional gel electrophoresis and mass spectrometry. Chromatin assembly factor 1 was uniquely expressed in pro-B cells. Purine nucleotide phosphorylase, LCK, E2A and many other unidentified proteins were dominantly present in the nucleus at the early stage of B cell development where the V(D)J recombination process occurs. Also, few proteins including guanidine nucleotide binding proteins were exclusively expressed in pre-B cell. Such findings can provide some information to help understand the developmental regulation of gene rearrangement occurring during B cell development.     

Regulation of the apoptotic response to radiation damage in B cell development

B lymphocyte precursor cells are ultrasensitive to DNA damage induced by irradiation and drugs and die by apoptosis at very low levels of exposure. Previous studies have shown that this high level sensitivity is p53-dependent, associated with very low level expression of Bcl-2 protein and can be reversed by expression of a bcl-2 transgene. We show here that transition from the pro-B to pre-B and then mature B cell stages of murine lymphopoiesis is accompanied by changes in proliferating cells in sensitivity to X-irradiation induced apoptosis and that this is paralleled by variation in the ratio of anti-(Bcl-2/Bcl-chiL) to pro-(Bax) apoptotic proteins. These are however not fixed or invariant features of developmental stage as they can be modulated by interactions via adhesive interactions with stromal cells, stromal proteins and growth factors. We interpret these data in the context of the stringent developmental regulation of clonal lymphopoiesis and the contingency programming of cells that have extensive proliferative potential with a very low threshold for apoptosis following DNA damage.     

The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors

The primary age-related loss in B cell progenitors is thought to be at the pro- to pre-B cell transition. However, we show that the frequencies and absolute numbers of all progenitor populations for the B cell lineage, including B-lineage-committed pro-B cells and multipotent B-lymphoid progenitors, decline in aged C57BL/6 mice. Moreover, when derived from aged mice, lymphoid progenitors within every population examined exhibited suboptimal IL-7 responsiveness, demonstrating that age-associated suboptimal IL-7R signaling is a general property of all early B-lineage precursors. Collectively, these data indicate that aging results in a previously unappreciated decline in the earliest stages of B cell development.     

CD19 function in early and late B cell development. II. CD19 facilitates the pro-B/pre-B transition

Proliferative expansion of pro-B cells is an IL-7-dependent process that allows for the rearrangement of H chain genes and the expression of the pre-B cell receptor (pre-BCR). Further B cell differentiation is dependent upon signals elicited through the pre-BCR, which are thought to be responsible for allelic exclusion, induced L chain gene rearrangement, and continued proliferation. CD19 promotes the proliferation and survival of mature B cells, but its role in early B cell development is less well understood. Here we identify and characterize impairments in early B cell development in CD19(-/-) mice. Following sublethal irradiation, we found decreased numbers of autoreconstituted early B cells, which was first evident in the large cycling pre-B cell fraction. Reduced cell progression due to a defect in proliferation was made evident from cell cycle analysis and bromodeoxyuridine labeling of bone marrow cells from CD19(-/-) and wild-type mice. Studies of IL-7-dependent pre-B cell cultures derived from wild-type and CD19(-/-) mouse bone marrow suggested that CD19 has little affect on IL-7 signaling. By contrast, signaling through the pre-BCR was impaired in the absence of CD19, as demonstrated by reduced activation of Bruton's tyrosine kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase. Thus, in addition to promoting mature B cell homeostasis and Ag-induced responses, the early onset of CD19 expression acts to enhance B cell generation.     

Proliferation of bone marrow pro-B cells is dependent on stimulation by the pituitary/thyroid axis

The frequency and absolute number of pro-B, pre-B, and B cells in the bone marrow of the hypothyroid strain of mice are significantly reduced compared with those of their normal littermates. To investigate why this is the case, various B cell developmental processes were examined in the thyroid hormone-deficient mice. These studies revealed that the frequency of pro-B cells in the S-G2/M phase of the cell cycle was significantly reduced in hypothyroid mice. That thyroid hormone deficiency was responsible for this proliferation defect was established by demonstrating that treatment of hypothyroid mice with thyroxine resulted in a specific increase in the frequency and total number of cycling pro-B cells. The latter effect was paralleled by increases in the frequency and number of bone marrow B lineage cells. Additional in vitro experiments revealed that at least some thyroid hormone effects were directly mediated on the bone marrow. Taken together, these data demonstrate that thyroid hormones are required for normal B cell production in the bone marrow through regulation of pro-B cell proliferation and establish a role for the pituitary/thyroid axis in B cell development.     

Expression of CD28 by bone marrow stromal cells and its involvement in B lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2-6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15-20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28(-/-) mice were similar to those in wild-type mice; however, older CD28(-/-) mice (15-19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.     

Analysis of VpreB expression during B lineage differentiation in lambda5-deficient mice

The VpreB/lambda5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of lambda5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in lambda5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.     

Reduced alpha4beta1 integrin/VCAM-1 interactions lead to impaired pre-B cell repopulation in alpha 1,6-fucosyltransferase deficient mice

Mice with a targeted gene disruption of Fut8 (Fut8(-/-)) showed an abnormality in the transition from pro-B cell to pre-B cell, reduced peripheral B cells, and a decreased immunoglobulin production. Alpha 1,6-fucosyltransferase (FUT8) is responsible for the alpha 1,6 core fucosylation of N-glycans, which could modify the functions of glycoproteins. The loss of a core fucose in both very late antigen 4 (VLA-4, alpha4beta1 integrin) and vascular cell adhesion molecule 1 (VCAM-1) led to a decreased binding between pre-B cells and stromal cells, which impaired pre-B cells generation in Fut8(-/-) mice. Moreover, the B lineage genes, such as CD79a, CD79b, Ebf1, and Tcfe2a, were downregulated in Fut8(-/-) pre-B cells. Indeed, the frequency of preBCR(+)CD79b(low) cells in bone marrow pre-B cells in Fut8(-/-) was much lower than that in Fut8(+/+) cells. These results reveal a new role of core fucosylated N-glycans in mediating early B cell development and functions.