Growth Of Lymphoid Cells Research Articles

Derivation of a new B-cell line from mouse spleen and its characterization

Prolonged culturing of splenocytes of (CBA×C57B1/6)F1 hybrid mice in RPMI-1640 medium with 15% fetal calf serum resulted in the derivation of a new B-cell line. After a monolayer of stromal fibroblast-like cells was formed, lymphoid cell growth was observed in 4–5 weeks of splenic cell culturing. All the cells of the splenic line belonged to the B series and expressed IgM on their surface; they did not form lymphoid colonies after injection to lethally irradiated mice, and even after 8-month culturing and several passages they could not be differentiated.

The interaction of (1-4)-fragment of thymosin beta 4 with calmodulin-sensitive cAMP phosphodiesterase from hypothalamus.

Evidence was accumulated indicating that cyclic nucleotides are involved in regulation of growth, differentiation and function of lymphoid cells. It was previously shown that the N-fragment (1-4) of thymosin beta 4 (Ac-Ser-Asp-Lys-Pro-OH) inhibits in vivo the entry of cell populations into S-phase. In the course of the study of the interrelationship between the immune and neuroendocrine systems we have found that the tetrapeptide caused incomplete competitive inhibition of hypothalamic calmodulin (CaM)-dependent phosphodiesterase (PDE) stimulated by CaM. In the presence of the peptide, the 20-fold increase of the constant for PDE activation by CaM was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis. The value of the inhibition constant (Ki) amounted to 600 nM. In the absence of CaM, the peptide at saturating concentrations reduced the basal activity of PDE nearly 2- to 3-fold. The effect of the peptide on PDE was noncompetitive with respect to cAMP. The results support our suggestion that the tetrapeptide realizes its effects in the immuno-neuroendocrine system by the mechanism of cyclic nucleotide metabolism.

CD27-CD70 interaction: unravelling its implication in normal and neoplastic B-cell growth.

Members of the Tumour Necrosis Factor-Receptor (TNFR) family play an essential role in the control of lymphoid cell growth and differentiation. The ligand of one of its lymphoid-specific members, CD27, was recently characterized as CD70, a type II transmembrane molecule with homology to TNF that is expressed on activated T and B cells. Ligation of CD27 by its natural ligand generates a potent costimulatory signal for cytokine production and proliferation of activated T cells. In contrast to normal B cells, where CD27 expression is confined to germinal centre cells and to a small subset of circulating B lymphocytes, CD27 expression is found on a large array of distinct B-cell neoplasia. Here, we review recent data on the expression and function of TNFR family members on normal and malignant lymphocytes and propose a role for CD27-CD70 interaction in B-cell development.

DNA-binding specificity and trans-activating potential of the leukemia-associated E2A-hepatic leukemia factor fusion protein.

Hybrid transcription factors, resulting from gene fusions in the wake of chromosomal translocations, have been implicated in leukemogenesis, but their precise contributions to oncogenic conversion remain unclear. The E2A-HLF fusion gene, formed by a t(17;19)(q22;p13) in childhood pro-B-cell acute lymphoid leukemia, encodes a hybrid protein that contains the trans-activation domain of E2A (E12/E47) linked to the bZIP DNA-binding and dimerization domain of hepatic leukemia factor (HLF). Here we report that both HLF and E2A-HLF bind to a 10-bp consensus sequence, 5'-GTTACGTAAT-3', with a core dyad-symmetric motif characteristic of the bZIP scissors-grip model of DNA binding. A probe containing this sequence bound chimeric E2A-HLF proteins in nuclear extracts of a leukemic cell line (UOC-B1) containing the t(17;19), as demonstrated by complexes supershifted with antibodies specific for amino-terminal epitopes of E2A or carboxyl-terminal eptiopes of HLF. E2A-HLF functioned as a potent trans activator of reporter gene expression from a plasmid that contained the consensus DNA-binding sequence. Interestingly, wild-type HLF was restricted in its capacity to act as a trans activator, functioning in human fetal kidney cells but not HepG2 hepatocarcinoma cells or NIH 3T3 mouse fibroblasts. The ability of the E2A-HLF hybrid protein to bind DNA in a sequence-specific manner and trans activate the expression of artificial reporter genes suggests that it could subvert transcriptional programs that normally control the growth, differentiation, and survival of lymphoid progenitor cells.

DNA-binding specificity and trans-activating potential of the leukemia-associated E2A-hepatic leukemia factor fusion protein.

Hybrid transcription factors, resulting from gene fusions in the wake of chromosomal translocations, have been implicated in leukemogenesis, but their precise contributions to oncogenic conversion remain unclear. The E2A-HLF fusion gene, formed by a t(17;19)(q22;p13) in childhood pro-B-cell acute lymphoid leukemia, encodes a hybrid protein that contains the trans-activation domain of E2A (E12/E47) linked to the bZIP DNA-binding and dimerization domain of hepatic leukemia factor (HLF). Here we report that both HLF and E2A-HLF bind to a 10-bp consensus sequence, 5'-GTTACGTAAT-3', with a core dyad-symmetric motif characteristic of the bZIP scissors-grip model of DNA binding. A probe containing this sequence bound chimeric E2A-HLF proteins in nuclear extracts of a leukemic cell line (UOC-B1) containing the t(17;19), as demonstrated by complexes supershifted with antibodies specific for amino-terminal epitopes of E2A or carboxyl-terminal eptiopes of HLF. E2A-HLF functioned as a potent trans activator of reporter gene expression from a plasmid that contained the consensus DNA-binding sequence. Interestingly, wild-type HLF was restricted in its capacity to act as a trans activator, functioning in human fetal kidney cells but not HepG2 hepatocarcinoma cells or NIH 3T3 mouse fibroblasts. The ability of the E2A-HLF hybrid protein to bind DNA in a sequence-specific manner and trans activate the expression of artificial reporter genes suggests that it could subvert transcriptional programs that normally control the growth, differentiation, and survival of lymphoid progenitor cells.

Oxysterol sensitive and resistant lymphoid cells: Correlation with regulation of cellular nucleic acid binding protein mRNA

Oxygenated derivatives of cholesterol inhibit cholesterol synthesis, prevent lymphoid cell growth, and evoke cell death. We have employed a novel selection method to isolate M10 cells, a line of oxysrerol-resistant cells, from the sensitive clone CEM C7. Concentrations of the potent sterol 25-hydroxycholesterol that occupy the oxysterol binding protein cause cell death in CEM C7, but not in M10 cells. Both cell lines have similar amounts of the oxysterol binding protein with similar affinities for oxysterol. However, in neither line are the levels of oxysterol binding protein mRNA affected by 1 μM 25-hydroxycholesterol. Furthermore, both cells express the cellular nucleic acid binding protein (CNBP), a 7 zinc finger, DNA-binding protein of unknown function, regulated by oxysterols. The levels of CNBP mRNA are significantly reduced by 25-hydroxycholesterol in the sensitive CEM C7 cells, in which the dose response and time course are consistent with occupancy of the oxysterol binding protein by oxysterol and with subsequent cell kill. However, in the resistant M10 cells, CNBP mRNA levels are unaffected by these concentrations of the 25-hydroxycholesterol. Our results suggest a role for CNBP in oxysterol-induced regulation of cell viability and growth.

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor.

Mice were injected in the foot pad with either 5 x 10(5) syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.

Hemopoietic functions of marrow-derived osteogenic cells.

Osteoblasts, members of the marrow stromal cellular network, may play an active role in the hemopoietic microenvironment as well as in bone remodeling. In this study, we examined the extent to which marrow-derived osteogenic cells (MBA-15) possess various stromal functions. This marrow stromal-derived cell line was shown by us to exhibit osteoblastic characteristics in culture and to form bone in vivo. These cells are shown here to constitutively produce and secrete cytokines identified as M-CSF, GM-CSF, and IL-6. MBA-15 cells modulate growth of normal and malignant myeloid and lymphoid cells as well as leukemia cell lines in vitro. Cell-cell interactions were studied in co-cultures with adherent MBA-15 cells and the target hemopoietic cells. Growth inhibition effects, observed under various experimental conditions, can be attributed to the presence of different soluble and membrane-bound inhibitory activities produced by MBA-15 cells. Thus, MBA-15 cells spontaneously produce both stimulators and inhibitors that can affect myeloid and lymphoid cell growth. Marrow osteogenic cells may therefore participate in the stromal regulation of hemopoiesis.

Effect of antiepileptic drugs on growth of murine lymphoid tumor cells in single-cell culture.

The effect of three commonly used antiepileptic drugs (AEDs), phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA), on the growth of lymphoid tumor cells was assessed in vitro. A single-cell culture method was used to determine growth rates by direct visualization. The amount of free drug was determined by ultrafiltration to ascertain its correlation to therapeutic drug levels. VPA slowed the growth of B-myeloma (FO) and T-lymphoma (AKR-1) cells significantly within the range of therapeutic drug levels. CBZ and PHT likewise inhibited cell growth in both lineages but at two to four times the therapeutic level of free drug. CBZ was shown to have long-term effects on FO and AKR-1 cells, demonstrated by the reduced growth rates of cloned lines for 2-3 months after drug removal. Cloned sublines of myeloma cells secreting lambda light chain (J558L) treated with CBZ or PHT had a higher frequency of lambda light chain secretion loss mutations than the nontreated parent line.

10 - Transforming growth factor β and interleukin-1: a paradigm for opposing regulation of haemopoiesis

The polypeptide cytokines, IL-1 and TGF-beta affect nearly every tissue and cell type in the body. IL-1 is the prototype of the proinflammatory molecule while TGF-beta is essentially anti-inflammatory. IL-1 is part of the cascade of cytokines that are produced during microbial invasion or bodily injury and enhance a variety of host responses, particularly in the immunological and haemopoietic systems, while TGF-beta acts as an inhibitor of these responses. At several levels, IL-1 and TGF-beta act in opposition to one another. IL-1 stimulates the expression of many genes in lymphoid and marrow stromal cells that stimulate haemopoietic cell growth and differentiation, while TGF-beta inhibits these IL-1 mediated effects. Also, TGF-beta stimulates secretion of the IL-1Ra. In addition, IL-1 induces the cell surface expression of cytokine receptors on lymphoid and haemopoietic cells, while TGF-beta dramatically inhibits the cell surface expression of these receptors, including the IL-1 receptor. Finally, IL-1 augments lymphoid and haemopoietic cell growth and TGF-beta potently inhibits this proliferation. The interactions of these cytokines serve to illustrate that the net balance of stimulatory and inhibitory signals determines the fate of a given cell which may be responsible for regulating homeostatic cell growth (Figure 1). Thus, the regulation of cytokine production and/or antagonism of their effects continues to be a therapeutic goal in the treatment of many diseases.

Hematopoietic growth factors: Current knowledge, future prospects

The introduction of hematopoietic growth factors into clinical medicine represents one of the more exciting developments in oncology in the past several years. The identification, gene cloning, and large-scale production of hematopoietic growth factors represent important examples of the practical benefits that may accrue from application of the sophisticated technology derived from recombinant DNA research. Research, both at the bench and by the bedside, has proceeded at an extraordinarily rapid pace in this field over the past five years, leading to an abundance of new information, novel promising agents, and important clinical controversies related to the biology and appropriate clinical applications of hematopoietic growth factors. With these agents, for the first time in history, the production of human blood cells can be systematically manipulated in vivo in an effort to optimize physiology beyond the endogenous host response. Additionally, investigators utilizing purified hematopoietic growth factors as reagents may provide crucial insights into the mechanisms of blood cell production in health and in various disease states. This review aims to summarize the current state of knowledge regarding the control of blood cell production by specific factors and to put these data in the context of clinical medicine. The emphasis will be on factors that primarily influence myeloid (rather than lymphoid) cell growth, differentiation, and activation, and the clinical focus will be on applications in oncologic therapeutics and in the treatment of primary hematologic disorders. By reviewing what we know and what has already been done, we may be better able to define the important questions that remain and to formulate the means to answer our current uncertainties about the activities and clinical uses of hematopoietic growth factors.

Interleukin-10

Despite the short history of interleukin-10, accumulated evidence indicates that this interleukin plays a major role in suppressing immune and inflammatory responses. Yet interleukin-10 also maintains cell viability and acts as a cofactor to promote the growth of lymphoid and myleloid cells in vitro. Here we review the present knowledge on the structure and function of interleukin-10.

C-fos gene induction by interleukin 2: identification of the critical cytoplasmic regions within the interleukin 2 receptor beta chain.

Interleukin 2 (IL-2) plays a critical role in the growth and differentiation of lymphoid cells. The IL-2 signal is delivered intracellularly by the IL-2 receptor beta chain (IL-2R beta); however, the mechanism by which the signal reaches the nucleus remains unclear. In this study, we demonstrate the rapid activation of c-fos protooncogene transcription by IL-2 and provide evidence that the serum-responsive element (SRE) within the c-fos promoter is responsible for the activation in a murine pro-B-cell line, BAF-B03, expressing the human IL-2R beta cDNA. Interestingly, the same SRE is also responsible for c-fos gene activation by interleukin 3 or erythropoietin. Further, we show that the activation of c-fos by IL-2 requires defined cytoplasmic regions of IL-2R beta--i.e., the "serine-rich" region, which is known to be essential for growth-signal transduction in BAF-B03 cells, and the "acidic region," which is located more distal to the cell membrane. These results indicate the functional importance of the two distinct regions within the IL-2R beta cytoplasmic domain in IL-2-induced c-fos gene activation and point to a potential role of the acidic region in IL-2 signal transduction that could not be adequately assessed in a previous study.

Stimulation of growth of human malignant lymphoma and lymphoid leukemia cells in vitro.

We have analyzed cultures of malignant lymphoma cells and cells from patients with acute lymphoid leukemia in methylcellulose for their ability to from colonies. Clonogenic growth was examined in the presence or absence of fetal calf serum (FCS), platelet-poor plasma (PPP), medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM), or irradiated allogeneic bone marrow stroma cells. Cells from 25 lymphoma patients--17 with non-Hodgkin's lymphoma (NHL), eight with Hodgkin's disease (HD)--and from 19 patients with acute lymphocytic leukemia (ALL) were investigated. We show that colony growth can be obtained in a minority of cases (in 3 NHL, 5 HD, and 2 ALL) and that the use of FCS and allogeneic irradiated stroma cells may be required for optimal colony formation.

Retinyl esters in chylomicron remnants inhibit growth of myeloid and lymphoid leukaemic cells

We have studied the effects of retinyl esters in chylomicron remnants on cell growth and differentiation of myeloid and lymphoid leukaemic cells. Ten mumol l-1 retinyl ester in chylomicron remnants effectively reduced proliferation of the myeloid leukaemic cell lines HL60, U937 and KG-1, and induced differentiation of 68% and 53% of the HL60 and U937 cells, respectively, in 5 days. While no effect on cell growth of the lymphoid cell lines Daudi, Raji and SOS was observed, 10 mumol 1-1 retinyl esters in chylomicron remnants reduced the growth of the B lymphoid cell line Reh by more than 50%. Primary cell cultures from six patients with acute leukaemia (four non-lymphocytic and two lymphocytic) were incubated with chylomicron remnant retinyl esters and proliferation was measured by means of thymidine incorporation. Among the myeloid leukaemic cells, the monomyelocytic, the two promyelocytic and the monoblastic leukaemic cells were growth inhibited. Chylomicron remnants had no effect on the growth of the c-ALL primary culture, but reduced proliferation of the T-ALL primary culture by approximately 20% after 48 h. These data suggest that high doses of retinol may be used in the treatment of some forms of acute leukaemia.

Prolactin receptor gene expression in lymphoid cells

To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.

The Clinical Potential of Recombinant Human Interleukin 4 and Alfa-2b Interferon

Recombinant human interleukin 4 and alfa-2b interferon (Intron A) exhibit multiple biological effects on cells of diverse origin. Interleukin 4 can affect the growth, differentiation and functional activity of lymphoid and myelomonocytic cells. Alfa-2b interferon expresses a broad spectrum of antiviral, immunomodulatory, and antiproliferative activities. These properties suggest a broad clinical application for both cytokines.

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.

Measurement of Soluble Interleukin 2 Receptor in Sera of Adult Patients with Hematological or Solid Malignancies.

Interleukin 2 (IL-2) exerts its biological activity through a specific membrane receptor. Using specific monoclonal antibodies directed against the IL-2 receptor, a soluble form of this receptor can be detected in the serum. This soluble part of the human IL-2 receptor (sIL-2R) is released by T and B lymphocytes and plays a role in lymphoid cell growth regulation. We have measured the sIL-2R by ELISA in the serum of patients with solid tumors and with hematological malignancies (44 chronic lymphocytic leukemias (CLL), 5 hairy cell leukemias (HCL), 14 Hodgkin's diseases (HKD), 34 non-Hodgkin's lymphomas (NHL) and 19 acute lymphoblastic leukemias (ALL). The mean sIL-2R level in 40 normal subjects was 173 + 168 U/ml (mean {pminus} SD). It was considerably increased in HCL: 17938 {pminus} 23748 (P < 0.0003). In CLL, a significant increase was found which was particularly pronounced at clinical stage III and IV with a mean level of 1299 {pminus} 1127 U/ml. In HKD, sIL-2R was slightly but significantly increased 519 + 524 U/ml (P < 0.0004). In NHL, the sIL-2R level was 1015 {pminus} 1022 U/ml, but this increase did not correlate with the clinical stage or the histological grade of the disease. In ALL, sIL-2R levels were also significantly increased 1633 + 1046 U/ml (P < 0.00001). In 69 patients with solid tumors (including lung carcinomas, gynecological and digestive malignancies), 39% of the patients tested had slightly increased sIL-2R levels. However, this increase, when present, could not be related to the tumor histology. These results suggest that sIL-2R measurement is a poor diagnostic marker for solid tumors and some lymphoproliferative disorders. However, in patients with hematological malignancies, it could be a useful tool to monitor lymphoid neoplasias.

Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.