Growth regulation of human B lymphocyte progenitor cells
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This thesis describes an investigation into the growth characteristics of a human pre-B cell acute lymphocytic leukaemia cell line, SMS-SB. Most lymphocytic tumours are difficult to adapt to tissue culture and enter a crisis after a few rounds of cell division where the majority of cells die. To sustain proliferation of the remaining cells, addition of exogenous mitogens is usually required. SMS-SB was an unusual leukaemia because the cells did not go through a crisis phase and grew indefinitely, in the absence of exogenous mitogens. This sustained proliferation in tissue culture appears to reflect the synthesis and secretion of an autocrine growth factor (s); the cells are density-dependent for growth, and proliferation can occur in media completely devoid of protein. The original aim of this work was to identify and characterise the autocrine growth factor, termed SB-AF. During investigations to identify cytokines with the ability to substitute for the autocrine growth factor activity, platelet-derived growth factor (PDGF) was shown to stimulate the growth of SMS-SB cells under low cell density conditions; SMS-SB cells are known to secrete PDGF and express PDGF receptors. However, antibody inhibition experiments suggest that PDGF cannot account for all the autocrine activity of SB-AF, thus other cytokine components of SB-AF were sought. CD23 is a 45kDa type-II transmembrane glycoprotein and a member of the C-type lectin superfamily. There is a soluble form of CD23 (sCD23) which is released by cleavage from the surface of cells into the extracellular fluid, and this form has been attributed multiple cytokine activities. It was discovered that sCD23 dramatically promotes thymidine incorporation by SMS-SB cells. The work of this thesis has shown that SMS-SB cells undergo apoptosis when cultured at low cell density; sCD23 is the only cytokine tested with the ability to prevent SMS-SB cell apoptosis. Apoptotic SMS-SB cells have low levels of the proto-oncogene bcl-2 but sCD23 can sustain bcl-2 levels in the cells. The investigations have shown that SMS-SB cells do not express CD23, negating the hypothesis that CD23 is acting in an autocrine fashion. The most interesting discovery made during these investigations was that SMS-SB cells bind CD23-containing liposomes specifically but they do not express the known receptors for CD23, namely CD21, CD11a and CD11b; SMS-SB cells express a novel CD23 receptor. Thus, SMS-SB cells express a novel receptor for CD23 and signalling via this receptor prevent apoptosis of the cells. Preliminary data is presented from CD23 affinity columns used to isolate and characterise the novel CD23 receptor. A protein of 85kDa has been identified as a candidate receptor, but further characterisation is required. SMS-SB cells will provide a good model to examine the role of autocrine growth factors in early B cell development, moreover, the discovery of a novel CD23 receptor on pre-B cells, implies a role for sCD23 in early B cell development. Since sCD23 has previously been shown to promote the growth and maturation of early T cells and myeloid progenitors, it will be interesting to investigate the role of CD23, and the novel receptor, in all aspects of haematopoiesis.Keywords:
Platelet-derived growth factor
CD23
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Signalling
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CD19, a B cell-specific transmembrane protein, is essential for murine B-1 cell development and T cell-dependent B cell immune responses. Whereas signaling by the human B cell Ag receptor can be modulated by CD19, less is known about the biochemical properties of murine CD19. We have used a novel rat mAb specific for murine CD19 to study the biochemical properties of the murine protein. We demonstrate that murine CD19 shares with human CD19 an association with complement receptor CD21 and CD81, tyrosine phosphorylation, binding of phosphatidylinositol-3 kinase, and synergistic signaling with membrane IgM. Murine CD19 is shown also to enhance signaling through the micro-surrogate light chain complex of primary pre-B cells. We found that although expressed in the earliest B cell precursors, CD19 ligation does not activate Ca2+ mobilization until the pre-B cell stage of development. In mature B cells, CD19 cross-linking activates Ca2+ flux in B-2 cells but not in B-1 cells, although it can synergize with surface IgM in both B-1 and B-2 cells. These biochemical properties of CD19 will be important for understanding its function in B cell development and the humoral immune response.
breakpoint cluster region
B-1 cell
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Abstract The importance of cytokines in controlling immunoglobulin isotype switching is well known. Given the defect in switching to IgG, IgA and IgE isotypes in mice and humans that carry mutations in the CD40 and CD40 ligand genes, we have investigated the role of CD40 ligation in controlling B cell responses to interleukin (IL)‐4. We have found that CD40‐mediated signals cause a fivefold up‐regulation of IL‐4 receptor (IL‐4R) on the B cell surface and that this is associated with a 100–1000‐fold increase in the cells' responsiveness to the cytokine. While we found no evidence of increased affinity or structural change of the receptor, we do find that prestimulation of B cells with anti‐CD40 antibodies brings about several changes in the IL‐4 signaling pathways. Subsequent delivery of IL‐4 to CD40‐prestimulated cells provokes intracellular signals distinct from those induced in resting B cells in response to IL‐4. While resting B cells phosphorylate Jak3 kinase shortly after IL‐4 activation, cells pre‐incubated with anti‐CD40 exhibit active dephosphorylation of this molecule and phosphorylation of proteins of around 45 kDa upon addition of IL‐4. The common γ chain, Jak3 and Jak1 can all be immunoprecipitated in normal amounts with the IL‐4R chain after CD40 prestimulation. We show that the observed dephosphorylation of Jak3 may be due to a stable association with the src ‐homology protein tyrosine phosphatase SH‐PTP2. In contrast, the enzyme appears to be inactive and to dissociate very quickly from the signaling complex in cells that are stimulated with IL‐4 alone.
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In the pathogenesis of atherosclerosis, circulating leukocytes adhere to endothelial cells, migrate through them and enter the vessel wall. CD40, a member of the tumor necrosis factor receptor family, that is expressed by both leukocytes and endothelial cells plays an important role in this process. Upon their emigration into the vessel wall leukocytes come into contact with the vascular smooth muscle cells (SMC). These cells also play a major role in the development and progression of atherosclerosis and express CD40 under pro-inflammatory conditions. The aim of the present study was to answer the question which role CD40 expression in SMC might play in the context of atherosclerosis. To address this complex question, four different experimental approaches were taken: (i) analysis of CD40 expression itself in human cultured SMC, (ii) analysis of CD154-induced gene expression in these cells, as well as (iii) the signal transduction pathways involved therein, and (iv) investigating possible functional consequences of these changes in gene expression. A first result achieved was that under pro-inflammatory conditions, CD40 expression is markedly up-regulated in human cultured SMC. Moreover, activation of CD40 in these cells resulted in the differential expression of 36 genes, as judged by DNA microarray and confirmed by RT-PCR analysis. Most of these gene products were up-regulated and comprised pro-inflammatory molecules, namely chemokines and their receptors, cytokines and their receptors, and adhesion molecules. The expression of these gene products in human SMC could be demonstrated for the first time. To elucidate the signalling pathways linking CD40-CD154 interaction to gene expression in the SMC, CD40-induced matrix metalloproteinase-3 (MMP-3) expression was chosen as a readout. By using different pharmacological inhibitors, it was demonstrated that the tyrosine kinase c-Src and the mitogen-activated protein kinase p38 are involved in CD40 signalling to the nucleus in these cells. Moreover, MMP-3 expression was verified to be up-regulated on the level of transcription, although the transcription factor(s) responsible therefor could not be identified as yet. By using two different experimental approaches, a SMC-monocyte interaction assay and analysing changes in gene expression in these cells upon exposure to the conditioned medium of CD154-stimulated SMC, it was further shown that monocyte activation is mediated by a humoral factor rather than a cell-to-cell contact. Further investigations, employing neutralizing antibodies, revealed that granulocyte-macrophage colony-stimulating factor is a likely candidate for the monocyte-activating humoral factor. Collectively, these findings suggest that vascular SMC like endothelial cells contribute to the maintenance of an inflammatory response in the vessel wall as, e.g., in atherosclerosis when stimulated via the CD40/CD154 receptor/ligand dyad. Elucidating the transcriptional mechanism by which CD40 activation in these cells is translated into the release of pro-inflammatory mediators represents a valuable therapeutic goal in this context.
CD154
Proinflammatory cytokine
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CD28 is a 44-kDa homodimeric receptor that is expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with phorbol ester (PMA) induces the production of cytokines and the proliferation of resting T cells via signal transduction pathways independent of the TCR. Evidence is provided herein that CD28 signals leading to cytokine production do not require the p59fyn (Fyn) tyrosine kinase, whereas CD28-mediated proliferation is dependent on the presence of the Fyn kinase in thymic, but not lymph node, cells. The defect in proliferation is not due to failure of IL-2R signaling, since addition of high concentrations of exogenous IL-2 can overcome the proliferative defect. Analysis of CD28-directed induction of the IL-2R alpha (CD25)-chain, which confers high affinity binding to IL-2, showed that Fyn-deficient thymocytes, but not lymph node cells, failed to up-regulate CD25 expression following anti-CD28 and PMA stimulation. Thus, the Fyn tyrosine kinase is critically required for thymic CD28-mediated CD25 expression and proliferation but not for CD28-mediated cytokine production.
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ZAP70
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Lymphocyte proliferation is stimulated by differential combinations of various cytokines, antigens and adhesion molecules. However, mechanisms of negative regulation in lymphocytes are poorly understood despite their potential importance in controlling the balance of lymphocyte proliferation, particularly at inflammatory sites. We recently reported a novel murine soluble protein, termed AIM, which inhibits apoptosis of a variety of cell types including CD4/CD8 double-positive thymocytes. AIM is secreted specifically by macrophages and belongs to the macrophage scavenger receptor cysteine-rich domain superfamily. Here we show that in addition to the apoptosis-inhibitory effect, AIM induces strong, long-term inhibition of B lymphocyte proliferation in combination with transforming growth factor-beta1 (TGF-beta1 in vitro), resulting in almost complete block of proliferation and immunoglobulin secretion. The function of AIM as a cell growth inhibitor requires pretreatment of B cells with TGF-beta1 which appears to increase expression of the AIM receptor on the B cell surface. Thus B lymphocyte proliferation is dramatically down-regulated by sequential exposition to TGF-beta1 followed by AIM. Like many cytokines, AIM has different functions depending on the types of target cells and the combination with other cytokines.
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Interleukin 3
Interleukin 15
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Engagement of the antigen receptor on WEHI 231 murine B lymphoma cells leads to growth arrest and induction of apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. At the molecular level, CD40 has been shown to activate nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK). The aim of our present study was to define the stretch of the CD40 cytoplasmic tail responsible for mediating these effects in WEHI 231 cells. Using recombinant retroviruses with the enhanced green fluorescent protein as selection marker we transduced WEHI 231 cells with chimeric molecules consisting of the extracellular and transmembrane region of human CD40 or rat CD4 and selected portions of the murine CD40 tail. Chimeric molecules with cytoplasmic fragments encompassing the "CD40 tumor necrosis factor-associated factor family member interacting motif" (TIM) were able to sustain growth and to uphold NF-kappaB activity as efficiently as the whole intracellular region of CD40. While the potential of the motif relative to the whole cytoplasmic tail was independent of the heterologous part of the chimeras it was strongly influenced by its distance to the membrane. Placing the 17-amino acid stretch of the motif too close to the membrane, i. e. only two or four amino acids apart, destroyed its capacity to mitigate the anti-IgM effect. Activation of SAPK through the chimeric molecules always correlated with their ability to activate NF-kappaB activity and to rescue the cells from apoptosis induced by antigen receptor ligation. Our data indicate that CD40-TIM carries most if not all of the information needed to deliver the signals responsible for sustaining growth in anti-IgM-stimulated WEHI 231 cells.
Chimera (genetics)
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The activation of B cells following encounter with antigen is tightly regulated during the course of a humoral immune response. The roles of factors produced by accessory cells, particularly T cells, are well established. However, the role of any B cell-derived factors is less clear, and it is possible that B cells regulate their own activation in an autocrine manner. The aim of this project was therefore to characterise the production and autocrine function of murine B cell-derived factors. B cells are observed to cluster upon activation in vitro, especially when stimulated via CD40, and B cell proliferation can be partially inhibited by blocking cell adhesion. It therefore appeared that B cell proximity was necessary for proliferation and this might have been due to the production of an autocrine growth factor. Here, a B cell autocrine growth factor activity was indeed demonstrated by separating cultures of B cells across dialysis tubing. This factor acted synergistically with CD40 stimulation. However, when B cells were cultured individually by embedding them in agarose, proliferation still occurred showing that B cell aggregation is not an absolute requirement for proliferation. An mRNA screen was performed in an attempt to identify cytokines produced by B cells upon activation. Several cytokine transcripts were discovered but none of these were strikingly upregulated following stimulation. Whilst tumour necrosis factor-α (TNF-α) has been reported to be a human B cell autocrine growth factor, murine B cells did not express TNF-α protein and TNF-α had no effect on murine B cell proliferation, activation or immunoglobulin secretion. Thus TNF-α is not an autocrine growth factor for murine B cells. Interleukin-6 was produced by murine B cells following activation, and this cytokine enhanced the survival of the B cells. Thus B cells clearly produce autocrine cytokines which regulate their activation.
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