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C J Paige

Publications and source records attributed to C J Paige.

At least 37 records · Page 2Linked to original sources
PubMed1996

Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities.

PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3-5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic-treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

Animals↗
PubMed1996

In vitro tracking of IL-7 responsiveness and gene expression during commitment of bipotent B-cell/macrophage progenitors.

BACKGROUND: The development of B lymphocytes from multipotent hematopoietic stem cells occurs through a series of intermediate cell types with increasingly restricted developmental potential. Despite intensive investigation, the underlying basis for commitment to a given lineage or the restriction in developmental potential of multipotent cells is unknown. To gain insight into this process we have developed an in vitro system that tracks a bipotent progenitor, which has the capacity to give rise to both B lymphocytes and macrophages, as it makes the transition to a B-lineage-committed precursor. The development of mature B lymphocytes from bipotent progenitors is dependent on interleukin 7 (IL-7), a pre-B-cell growth factor, in addition to other stromal-cell-derived factors such as IL-11 and mast cell growth factor (MGF). IL-7 acts on pre-B lymphocytes, but the stage of differentiation at which B-lineage cells become responsive to this factor, and its potential role in lineage commitment have not been investigated thoroughly. Here, we examine the requirements for IL-7 during the development of B lymphocytes from bipotent progenitors. Furthermore, we define onset of B-lineage-associated gene expression during the development of committed B-lineage cells under defined culture conditions. RESULTS: We demonstrate that, under our experimental conditions, bipotent progenitors commit to differentiation through either the B or macrophage lineages within the first 3 days of culture. Cells that require IL-7 for survival first develop on day 3 of culture; however, commitment to the B lineage occurs at the same frequency in the presence or absence of this factor. After day 3 of culture, IL-7 is required both for the proliferation and survival of committed B-lineage progenitors and for the expression of several B-cell-associated genes, such as lambda5, VpreB, mb-1 and Rag1. CONCLUSIONS: Our results demonstrate that the growth factor combination of IL-11 and MGF provides sufficient support for bipotent progenitors to commit to either the B or the macrophage lineage. Single-cell cloning assays revealed that IL-7 does not influence the decision to commit to the B lineage, despite the observation that the bipotent cells potentially respond to IL-7, as indicated by an increase in cell number, prior to the commitment event. Furthermore, the addition of IL-7 to cells developing along the B-cell pathway promotes the expression of mRNA transcripts which encode several B-cell-specific genes.

Animals↗
PubMed1996

Immunoglobulin-mediated signal transduction in B cells from CD45-deficient mice.

CD45 expression is essential for immunoglobulin (Ig)-mediated B cell activation. Treatments with either anti-Ig or anti-CD45 suggest that CD45 may facilitate early signaling events such as calcium mobilization, and phosphoinositide hydrolyis as well as later events leading to transcription of genes such as c-myc. To examine the role of CD45 more extensively, CD45-deficient mice were generated by disruption of exon 6. Although normal numbers of B cells were found in peripheral lymphoid tissues, CD45-deficient cells failed to proliferate upon IgM crosslinking. In the present study, we demonstrate that the fraction of high buoyant density B cells is reduced while low buoyant density cells are increased. Moreover, there is a significant decline in the number of splenic B cells of the mature IgDhi, IgMlo phenotype. Although both the basal and anti-Ig-induced levels of phosphorylation of Ig-alpha and phospholipase C gamma 2 are indistinguishable from that observed in CD45+ control B cells, a major distinction was found in Ca2+ mobilization. While anti-Ig-induced mobilization of intracellular Ca2+ stores was normal, influx from extracellular sources was abrogated. This finding reveals a novel pathway of regulating B cell responses mediated by CD45.

Animals↗
PubMed1996

Characterization of thymic stromal-derived lymphopoietin (TSLP) in murine B cell development in vitro.

B cell development is dependent on both direct interactions with stromal cells and their secreted cytokines. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. We report here that a novel growth factor thymic stromal-derived lymphopoietin (TSLP) can replace the activity of interleukin-7 (IL-7) in supporting B cell development in vitro. TSLP was found to promote the proliferation and differentiation of committed B220+ B cell progenitors from day 15 fetal liver. Phenotypic analysis of these cells revealed that they are at the pro-B cell stage of differentiation and express cell surface markers characteristic of pro-B cells cultured in IL-7. TSLP can replace the activity of IL-7 in supporting the progression of B lymphocytes from uncommitted bipotential precursors. In the absence of either TSLP or IL-7, the progeny of cells that give rise to mature B lymphocytes fail to develop from these bipotential precursors. Moreover, TSLP can substitute for IL-7 in supporting the sustained proliferative response exhibited by B cell progenitors from CBA/N mice. Together these results show that TSLP can replace the requirement for IL-7 during in vitro B cell development.

Animals↗
PubMed1996

Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7.

B cell development is influenced by interactions between B cell progenitors and stromal cells. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. Flt3 ligand (FL) is a growth factor which stimulates the proliferation of stem cells and early progenitors. Mice deficient for the FLT3 receptor exhibit severe reductions in early B lymphoid progenitors. We have previously described a clonal assay in vitro which allows us to follow the entire B cell differentiation pathway from uncommitted progenitors to mature, immunoglobulin-secreting plasma cells. The growth factor combination of interleukin (IL)-11, mast cell growth factor (MGF) and IL-7 was shown to maintain the differentiation of these hematopoietic precursors into B cell progenitors capable of giving rise to functionally mature B cells in secondary cultures. Here, we show that FL in combination with IL-11 and IL-7 is sufficient to support the differentiation of uncommitted progenitors from day 10 yolk sac (AA4.1+) or day 12 fetal liver (AA4.1+ B220- Mac-1- Sca-1+) into the B lineage. The frequency of B cell progenitors obtained in these conditions was similar, if not better, than the frequency of B cell precursors that arose when cultured in IL-11+MGF+IL-7. Furthermore, the growth factor combination of IL-11+FL+ IL-7 was able to maintain the potential of bipotent precursors giving rise to both the B and myeloid lineages in secondary cultures. We also show that FL synergizes with IL-7 in the proliferation of committed B220+ pro-B cells and may contribute to the maintenance of an earlier pro-B cell population. Together, these results show that FL is important in supporting the differentiation and proliferation of early B cell progenitors in vitro.

Animals↗
PubMed1995

Characterization of the 3' untranslated region of the mouse homeobox gene HoxB5.

The mouse pre-B cell line, 70Z/3, expresses multiple transcripts of the homeobox gene, HoxB5. We show here that this heterogeneity is due, at least in part, to the usage of alternative poly-A addition sites in the 3' untranslated region (UT) of the primary HoxB5 transcript. Furthermore, upon analysis of the subcellular distribution of the different HoxB5 RNA species, we found that the transcripts are present mainly in the nucleus, with two-to-five-fold less RNA present in the cytoplasm. These studies suggest that multiple post-transcriptional regulatory mechanisms are involved in the expression of HoxB5 RNA.

Animals↗
PubMed1995

Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta chain-independent pathway.

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.

Animals↗
PubMed1995

In-vitro models of B-lineage commitment.

The development of mature B lymphocytes from multipotent progenitors follows a pathway of differentiation marked by a progressive restriction in lineage options. The requirements for progression through the B lineage developmental pathway have been investigated intensively and a number of critical components of the differentiation process have been identified. However, the genetic basis for lineage determination remains unresolved. Recently, a number of in-vitro assays have been established which support the development of committed B cell progenitors from multipotent cells. These assays have provided a novel system in which the process of B lineage commitment can be followed and manipulated. In this review we present a model of B-lineage progression from multipotent progenitors to committed B-cell progenitors and discuss potential mediators of the commitment process.

Animals↗
PubMed1995

Murine B cell development: commitment and progression from multipotential progenitors to mature B lymphocytes.

B lymphocytes, the cellular source of antibody, are critical components of the immune response. They develop from multipotential stem cells, progressively acquiring the traits that allow them to function as mature B lymphocytes. This developmental program is dependent on appropriate interactions with the surrounding environment. These interactions, mediated by cell-cell and cell-matrix interactions, provide the growth and differentiation signals that promote progression along the developmental pathway. This chapter addresses the properties of developing B lineage cells and the nature of the environmental signals that support B lineage progression.

Animals↗
PubMed1994

Leukocyte common antigen (CD45) is required for immunoglobulin E-mediated degranulation of mast cells.

We demonstrate using primary mast cell cultures derived from wild-type and CD45-deficient mice that mast cell triggering through the high-affinity immunoglobulin E (IgE) receptor requires the cell surface tyrosine phosphatase CD45. Unlike wild-type cells, cross-linking of surface-bound IgE in mast cells deficient in CD45 does not induce degranulation. Degranulation in these mutant cells does occur after treatment with the calcium ionophore A23187 indicating that the degranulation machinery is intact in these cells. We also demonstrate that the tyrosine phosphatase inhibitors orthoVanadate and perVanadate inhibit degranulation in wild-type mast cells, as does cross-linking of CD45 by anti-CD45 antibodies. Finally, we show that CD45-deficient mice are resistant to IgE-dependent systemic anaphylaxis. These results show that, like the T cell receptor and the antigen receptor on B cells, there is an absolute requirement for CD45 in signaling via the high affinity IgE receptor, expanding the number of receptors for which CD45 is an essential component.

Anaphylaxis↗
PubMed1994

Kappa light chain rearrangement in mouse fetal liver.

Ig variable domains are generated by the recombination of V, D, and J segments (V(D)J rearrangement). V(D)J rearrangement is capable of generating a vast repertoire of different variable domains. In this report, we quantify and characterize the repertoire of kappa rearrangements in fetal liver ontogeny. VJ kappa rearrangements are first observable at approximately day 14 of gestation. Characterization of these rearrangements indicates that only 33% are in a productive reading frame, which supports the argument that they have been generated recently and have not as yet undergone significant Ag-driven selection. Further analysis of rearrangements from a pool of 133 cloned VJ kappa junctions (from both day 14 and day 16 of gestation) indicates that the repertoire is fairly diverse with respect to the V kappa gene families used, as well as the number of members from each gene family. The frequency of V kappa 4 family use in rearrangements to J kappa 5, however, was approximately twice that of the frequency of V kappa 4 family use in rearrangement to other J kappa s. The fine structure of fetal VJ kappa junctions was also diverse, which indicates that precise deletion of sequence identities shared between rearranging V kappa J kappa pairs does not significantly reduce junctional diversity, as has been observed in DJH rearrangements. Lastly, a number of junctions contained P nucleotides, in contrast to the repertoire of expressed VJ kappa junctions.

Animals↗
PubMed1994

Characterization of the B cell-macrophage lineage transition in 70Z/3 cells.

The 70Z/3 cell line is able to undergo lineage conversion from a pre-B to macrophage phenotype. Data presented here show that the transition from pre-B to macrophage follows a reproducible pathway via a stable intermediate stage. The cells in the intermediate stage are adherent and have lost the ability to respond to lipopolysaccharide or interferon-gamma by induction of immunoglobulin kappa light chains. However, these cells do not yet display the full range of macrophage-specific properties such as receptors for macrophage-colony stimulating factor or the beta 2 integrin CD11b/CD18. Subcloning experiments with the intermediate cells revealed that they retain the options of either persisting along the macrophage line of differentiation, acquiring additional macrophage traits, or reverting to the pre-B phenotype. Further differentiation to the macrophage stage is accompanied by the apparent loss of the ability to revert. Thus, these studies define relationships among lineage-specific traits, and begin to reveal critical stages in lineage commitment.

Animals↗
PubMed1994

Class II major histocompatibility complex-restricted T cell function in CD4-deficient mice.

Previously, we and others have demonstrated that CD4-deficient mice have a normal number of T cells and B cells with a significant population of CD4-8-TcR alpha beta+ T cells. Surprisingly, however, these mice lacking CD4 show in vivo immunoglobulin isotype class switching from IgM to IgG in response to sheep erythrocytes and vesicular stomatitis virus. In this study we have depleted various subpopulations of T cells in vivo and shown that the population of CD4-8-TcR alpha beta+ T cells is responsible for providing "help" in the antibody response of CD4-deficient mice to vesicular stomatitis virus infection. We have used antigen-specific proliferation assays and blocking studies with class I and II major histocompatibility complex (MHC)-specific purified antibodies to show that these cells are class II MHC-restricted in responses against the T cell-dependent antigen keyhole limpet hemocyanin (KLH). Finally, phenotypic analysis of the CD4-CD8- thymocytes in CD4-deficient mice shows that these cells have a more mature phenotype than the CD4-8- thymocytes in wild type mice. These results indicate that CD4 is not absolutely necessary for positive selection or effector function of class II MHC-restricted helper T cells.

Animals↗
PubMed1994

Stromal cell independent growth of bipotent B cell--macrophage precursors from murine fetal liver.

We have established stromal cell independent culture conditions under which single murine hematopoietic progenitor cells give rise to B lymphocytes and macrophages. IL-11, mast cell growth factor, and IL-7 are sufficient for the growth of B lineage cells from AA4.1+B220-Mac-1-Ly6A+ fetal liver cells isolated at day 12 of gestation. These conditions are also sufficient for commitment to myeloid differentiation, although CSF-1 is required for substantial growth of macrophages. The progenitors responding under these culture conditions: (i) require 8 days in culture before they become competent to respond to B cell mitogens in the presence of stromal cells and (ii) undergo a minimum of three cell divisions prior to Ig heavy chain allotype restriction. The findings suggest that the stromal cell dependency of immature hematopoietic cells may reflect a requirement for stromal cell derived growth factors. Further, this provides an in vitro system in which commitment and differentiation can be studied in single cells.

Animals↗
PubMed1994

Overusage of mouse DH gene segment, DFL16.1, is strain-dependent and determined by cis-acting elements.

The DJH structure is of particular importance for diversity in the immunoglobulin heavy chain because it encodes most of CDR3. Here, we investigate mechanisms responsible for generating the DJH structure. We found DFL16.1 was used at a high frequency in normal and transformed pre-B cells (fetal liver > 50%, A-MuLV lines approximately equal to 25%). One DFL16.1JH1 structure was found repeatedly and was also present in DJH and VDJH databases, suggesting this structure may be conserved in the primary repertoire. Genetic analysis demonstrated that C57BL/6 mice use DFL16.1 in DJH structures more frequently than BALB/c. Examination of individual alleles in (C57BL/6 x BALB/c)F1 A-MuLV cell lines revealed that the C57BL/6-derived allele used DFL16.1 twice as often as the BALB/c. This result indicates that part of the mechanism ensuring overusage of DFL16.1 gene segments is cis-acting.

Alleles↗
PubMed1993

Differentiation and characterization of B-cell precursors detected in the yolk sac and embryo body of embryos beginning at the 10- to 12-somite stage.

The embryonic sites in which progenitors of the hematopoietic lineages first emerge are ideal regions to characterize both the cells and environment needed to initiate blood cell development. For a number of years both the murine yolk sac and embryo have been recognized to contain progenitors of B lymphocytes. However, clonal, quantitative in vitro assays, which allow precise observation of precursors and their progeny, have been lacking. Moreover, the site of origin of the initial events remains controversial. In this report we document the presence of B-cell progenitors in yolk sac and embryonic tissue obtained from mouse fetuses beginning at the 10-somite stage, day 8.5. We determine the frequency, cell-surface phenotype, and growth properties of these progenitors. We show that these cells can differentiate into immunoglobulin-secreting cells and that the progeny derived from single progenitors are diverse with respect to immunoglobulin heavy-chain allotype expression, diversity-joining region use, and heavy-chain variable-region utilization.

Animals↗