Unlinked regulation of cell growth and differentiation in immature B cell lines

We established many immunoglobulin-null immature B cell lines transformed by tsOS-59, a temperature-sensitive mutant of Abelson murine leukemia virus. In different cell lines cell growth was depressed and cell differentiation (generation of intracytoplasmic mu-positive cells from Ig- cells) was induced by the shift of culture temperature from low (35 degrees C) to high (39 degrees C). Cell lines were categorized into four groups: (i) temperature sensitive (ts) to both cell growth and differentiation, (ii) ts to cell growth but not to cell differentiation, (iii) ts to cell differentiation but not to cell growth, and (iv) ts to neither cell growth nor differentiation. These results indicated that the depression of cell growth did not necessarily induce cell differentiation, and that cell differentiation was induced regardless of whether cell growth was depressed or not. Furthermore, the results showed that the depression of cell growth and the induction of cell differentiation occurred without the reduction of tyrosine kinase activity of P120gag-abl at high, nonpermissive temperature. Our cell growth and differentiation system described here should provide us with the interesting findings of the relation between B cell growth and differentiation.     

Human B cell growth and differentiation in the spleen of immunodeficient mice

Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor beta-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.     

Cooperation between soluble factors and integrin-mediated cell anchorage in the control of cell growth and differentiation

Recently it has become clear that integrins and other adhesive receptors play an important role in the control of cell growth and differentiation. In various cell types, anchorage to the extracellular matrix via integrins strongly influences the ability of the cell to respond to soluble mitogens or to differentiation factors. Thus adhesive receptors must generate signals that influence cell behavior. Some of the pathways of adhesion receptor signaling are now beginning to be worked out, but there is still much to learn. In particular, the mechanistic basis for the cooperation between anchorage signals and signals from soluble growth and differentiation factors remains ill-defined. This review will examine some of the current information linking adhesion receptors to control of mitogenesis and differentiation.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Ligand binding sites for a synthetic B cell growth and differentiation factor

The mechanism of action of a group of synthetic lymphokine-like molecules, the C8-substituted guanine ribonucleosides, was studied. Among their pleiotropic effects on B cells are the increased expression of surface Ia antigens, induction of polyclonal immunoglobulin secretion, enhancement of thymus-dependent as well as thymus-independent antibody responses, and transmission of T cell-like differentiative signals to B cells. However, relatively little is known about their molecular mechanism of action. In the current article, the interaction of 8-bromo-guanosine (8BrGuo), a prototypical C8-substituted guanine ribonucleoside, with cellular components was examined. Rapidly exchangeable (free) and slowly exchangeable (bound) 8BrGuo pools exist within B cells. The bound nucleoside pool loses its ability to be retained by a boronate affinity resin (despite its resistance to metabolic processing) and localizes to the cytosol on sucrose density gradients. Binding affinity, ligand specificity, and cellular specificity of binding all correlate closely with observed functional properties of these molecules. Together, these data suggest that the binding interaction mediates the biologic activities of 8BrGuo, and that the binding site acts as a functional nucleoside receptor.     

Lymphoma models for B cell activation and tolerance. I. Conditions for the anti-mu-dependent stimulation of growth in NBL, a nude B cell lymphoma

NBL is a spontaneous B cell lymphoma that originated in NIH Swiss nude mouse, and has been maintained as an in vitro line for 4 yr in our laboratory. It is surface IgM positive and expresses several B cell markers including Fc receptors, as well as Ly-1. Although clones of NBL will grow in serum-containing medium, this cell line enters a quiescent state in serum-free culture. However, in the presence of affinity-purified or monoclonal anti-mu reagents, NBL increases its rate of proliferation as measured by thymidine incorporation or in absolute cell numbers. This stimulation is specific for mu-chain, because it does not occur with anti-beta 2 microglobulin, irrelevant nonbinding antibodies, or with monoclonal anti-B cell lineage markers. Bivalent anti-mu is required, and no consistent Fc-mediated inhibition of growth has been detected. Stimulation of NBL occurs optimally at critical cell densities (greater than 3 X 10(3)/well) in the absence of serum. Therefore, we reasoned that NBL either produced or was receptive to known B cell growth factors. Although no classic IL 1 was detected in NBL supernatants, some BCGF-I-like activity was found. Finally, in the presence of LPS, both spontaneous and anti-mu-stimulated NBL growth was inhibited, a result suggesting maturation of this lymphoma. These results suggest that NBL represents an excellent model to study the growth and differentiation of B cell subsets.     

Interferons and oncogenes in the control of cell growth and differentiation: working hypothesis and experimental facts

This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.     

Idiotypic regulation of B cell differentiation

We study the equilibrium properties of idiotypically interacting B cell clones in the case where only the differentiation of B cells is affected by idiotypic interactions. Furthermore, we assume that clones may recognize and be stimulated by self antigen in the same fashion as by antiantibodies. For idiotypically interacting pairs of non-autoreactive clones we observe three qualitatively different dynamical regimes. In the first regime, at small antibody production an antibody-free fixed point, the virgin state, is the only attractor of the system. For intermediate antibody production, a symmetric activated state replaces the virgin state as the only attractor of the system. For large antibody production, finally, the symmetric activated state gives way to two asymmetric activated states where one clone suppresses the other clone. If one or both clones in the pair are autoreactive there is no virgin state. However, we still observe the switch from an almost symmetric activated state to two asymmetric activated states. The two asymmetric activated states at high antibody production have profoundly different implications for a self antigen which is recognized by one of the clones of the pair. In the attractor characterized by high autoantibody concentration the self antigen is attacked vigorously by the immune system while in the opposite steady state the tiny amount of autoantibody hardly affects the self antigen. Accordingly, we call the first state the autoimmune state and the second the tolerant state. In the tolerant state the autoreactive clone is down-regulated by its anti-idiotype providing an efficient mechanism to prevent an autoimmune reaction. However, the antibody production required to achieve this anti-idiotypic control of autoantibodies is rather large.     

Constitutive production by the WEHI-3 cell line of B cell growth and differentiation factor that co-purifies with interleukin 1

The myelomonocytic cell line WEHI-3 produces constitutively a factor that affects the growth and differentiation of murine B cells in culture. This cell line also secretes colony-stimulating factors (CSF), interleukin 1 (IL 1) but not interleukin 2. Sequential purification through AcA54 gel filtration, DEAE-Sephacel ion exchange chromatography, and buffer electrofocussing clearly resolved the B cell growth and differentiation factor (BGDF) from the CSF activities but failed to separate BGDF from IL 1. The WEHI-3-derived material responsible for BGDF/IL 1 activity, however, exhibited different behavior on DEAE chromatography (elution at 175 mM NaCl) to that reported for IL 1 from the P388D1 cell line (elution at 50 mM NaCl). B cell growth and differentiation could be induced by WEHI-3 BGDF/IL 1 in cultures of normal spleen cells depleted of T cells and adherent cells but not in cultures of spleen cells from B cell-deficient CBA/N mice, even though thymocytes from such mice displayed a normal response to IL 1. Significant B cell proliferation induced by BGDF/IL 1 was apparent only in the presence of submitogenic concentrations of anti-mouse IgM antibodies, but under these conditions few antibody-forming cells (AFC) were generated. In contrast, B cell differentiation to AFC occurred in the presence of the factor alone, and this response was inhibited by anti-IgM. Thus there appeared to be a reciprocal relationship between B cell proliferation and differentiation induced by BGDF/IL 1. The significance of these results is discussed in the light of other recent studies of BGDF.     

Hormonal control of leydig cell differentiation

Summary The fine structure of the testicular interstitial cells of the 9-day-old mouse submitted to stimulation with human chorionic gonadotropin (HCG) is reported. As was previously described (Baillie 1964) the interstitial tissue of the prepubertal mouse testis is characterized by the presence of well differentiated epithelioid cells at a quiescent stage. They are characterized by large cytoplasmic depots of lipid droplets and glycogen particles in contrast to poorly developed membranous organelles. These cells are highly sensitive to the action of gonadotropins. Five daily injections of HCG cause their differentiation into cells with active secretory characteristics. The gonadotropin induces a marked depletion of the lipid droplets and glycogen content of the cytoplasm, concurrent with an unusual development of the membranes of the agranular endoplasmic reticulum. Golgi complexes and rough reticulum are prominent. Several changes also appear in the nucleus, especially in the nucleolus.The correlation of the present electron microscopic study of the interstitial cells under HCG stimulation with previous biochemical and physiological findings tentatively suggests that the immature Leydig cells exhibit the basic organization necessary for biosynthesis of steroid hormones.     

Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks

Development of characteristic tissue patterns requires that individual cells be switched locally between different phenotypes or "fates;" while one cell may proliferate, its neighbors may differentiate or die. Recent studies have revealed that local switching between these different gene programs is controlled through interplay between soluble growth factors, insoluble extracellular matrix molecules, and mechanical forces which produce cell shape distortion. Although the precise molecular basis remains unknown, shape-dependent control of cell growth and function appears to be mediated by tension-dependent changes in the actin cytoskeleton. However, the question remains: how can a generalized physical stimulus, such as cell distortion, activate the same set of genes and signaling proteins that are triggered by molecules which bind to specific cell surface receptors. In this article, we use computer simulations based on dynamic Boolean networks to show that the different cell fates that a particular cell can exhibit may represent a preprogrammed set of common end programs or "attractors" which self-organize within the cell's regulatory networks. In this type of dynamic network model of information processing, generalized stimuli (e.g., mechanical forces) and specific molecular cues elicit signals which follow different trajectories, but eventually converge onto one of a small set of common end programs (growth, quiescence, differentiation, apoptosis, etc.). In other words, if cells use this type of information processing system, then control of cell function would involve selection of preexisting (latent) behavioral modes of the cell, rather than instruction by specific binding molecules. Importantly, the results of the computer simulation closely mimic experimental data obtained with living endothelial cells. The major implication of this finding is that current methods used for analysis of cell function that rely on characterization of linear signaling pathways or clusters of genes with common activity profiles may overlook the most critical features of cellular information processing which normally determine how signal specificity is established and maintained in living cells.     

Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation

Experimental evidence is presented that supports a cell cycle model showing that there are five distinct biological processes involved in proadipocyte differentiation. These include: (a) growth arrest at a distinct state in the G1 phase of the cell cycle; (b) nonterminal differentiation; (c) terminal differentiation; (d) loss of the differentiated phenotype; and (e) reinitiation of cell proliferation. Each of these events is shown to be regulated by specific human plasma components or other physiological factors. At two states designated GD and GD', coupling of growth arrest and differentiation is shown to occur. We propose that these mechanisms for the coupling of growth arrest and differentiation are physiologically significant and mimic the regulatory processes that control stem cell proliferation in vivo.     

Investigation of systemic control of plant cell division and differentiation in the model of tumor growth in radish

The study addresses the control of plant cell division and differentiation using the model of tumor-forming lines of radish. Expression of the genes involved in control of the cell cycle (CycD3), maintenance of meristematic cell activity (STM, WUS, and KNAT1), and primary response to cytokinin (ARR) was studied in inbred radish lines characterized by tumor growth at different stages of development. The influence of exogenic cytokinin on the expression of the genes of interest is analyzed. The possible role of the CycD3, KNAT1, STM, WUS, and ARR5 in tumor formation in radish is discussed.     

Investigation of systemic control of plant cell division and differentiation in the model of tumor growth in radish

The study addresses the control of plant cell division and differentiation using the model of tumor-forming lines of radish. Expression of the genes involved in control of the cell cycle (CycD3), maintenance of meristematic cell activity (STM, WUS, and KNAT1), and primary response to cytokinin (ARR) was studied in inbred radish lines characterized by tumor growth at different stages of development. The influence of exogenic cytokinin on the expression of the genes of interest is analyzed. The possible role of the CycD3, KNAT1, tSTM, WUS, and ARR5 in tumor formation in radish is discussed.     

Interferons and cell growth control

Cytokines modulate cell growth, differentiation, and immune defenses in the vertebrates. Interferons (IFNs) are a unique class of cytokines that stimulate antiviral, antitumor and antigen presentation by inducing the expression of several cellular genes. Recent studies have identified a novel gene regulatory pathway activated by IFNs, which serves as a paradigm for most cytokine signal transduction pathways. A number of genes induced by IFNs participate in cell growth regulation and apoptosis. These include novel tumor suppressor genes. Although discovered as IFN-regulated factors, deletions of these genes cause leukemias in experimental models and in human patients. Genetic approaches have identified several novel regulators of apoptosis. Studies on the mechanism of action of these growth regulatory molecules are not only useful in identifying novel targets for the development of therapeutics but also help understand the molecular basis for loss of cell growth control and resistance to IFNs. This review focuses on the functions and roles of IFN regulated factors in cell growth control and mechanisms of disruption of IFN action in cancer cells.     

Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells

Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported pI 5 for IL-6 and more acidic than the documented basic pI of IFN-gamma. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pI 6 BCDF, suggesting non-identity between IL-6 and anti-CD3 induced BCDF. Thus, anti-CD3 activated T cells generate both growth factor activity and BCDF as separate molecular entities distinct from IFN-gamma, IL-2, IL-4, and conventional IL-6.