Several types of adherent cells elaborate a dialyzable lymphopoietic cofactor (Abelson Growth Promoter)

We have cloned a stromal cell from mouse bone marrow selected on the basis of its ability to promote the growth of an Abelson virus-transformed pre-B cell line. These stromal cells have smooth muscle features, and the ultrafiltrate of stromal cell-conditioned medium has proliferative effects on all feeder layer-responsive mouse pre-B cell lines tested, as well as on normal pre-B cells. One of these low MW substances is a protease-resistant factor with an MW of approximately 450 Da which we designate Abelson Growth Promoter (AGP). AGP was initially characterized as an activity which promotes the growth of Abelson virus-transformed mouse pre-B cells, but it also promotes the growth of a ras-transformed pre-B cell line as a single agent and in synergistic fashion with recombinant interleukin (IL) 7. AGP as a single agent has no effect on normal pre-B cells, which have a brisk response to IL-7. In contrast, transformed pre-B cells display a blunted response to IL-7. We propose that AGP plays a role in normal lymphopoiesis by expanding clones of pre-B cells which have been activated by other stromal cell-derived signals, such as IL-7, and directly promotes the growth of nascently transformed pre-B cells.     

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.     

Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.     

The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7.

The cDNA for stem cell factor was recently isolated from Buffalo rat liver cells (BRL-3A) and recombinant rat stem cell factor produced from Escherichia coli (rrSCF164). rrSCF164 synergizes with rhIL-7 to stimulate pre-B clonal growth in agar culture of mouse bone marrow cells, and in this study we have characterized the role of rrSCF164 in B cell development. The combination of rrSCF164 plus rhIL-7 stimulated increased colony numbers compared with the sum of colonies stimulated by rrSCF164 and rhIL-7 alone. Also, increased cell proliferation per colony was stimulated by the combination of rrSCF164 plus rhIL-7 compared with rhIL-7 or rrSCF164 alone. The colonies formed with rrSCF164 plus rhIL-7 and rhIL-7 alone contained exclusively pre-B cells, which expressed B220 Ag and cytoplasmic mu-chain, but were negative for surface Ig expression. Morphological examination of the cells in the colonies showed blast-like characteristics. rrSCF164 alone and in combination with rhIL-7 stimulated generation of B220+ cells in liquid culture of B220- cells, whereas rhIL-7 alone had no stimulatory effect on B220- cells. Both stem cell factor mRNA and bioactivity were detected in a mouse bone marrow-derived stromal cell line, termed OZ-11. We propose that stem cell factor is a stromal-derived factor that synergizes with IL-7 to stimulate the proliferation and differentiation of pro-B cells to pre-B cells, which become responsive to IL-7 alone.     

IL-4 regulates differentiation and proliferation of human precursor B cells

The mechanism by which precursor and pre-B cells undergo differentiation is unclear; however, it is known that growth factors play an important role in this maturation process. The lymphokine, IL-4 has been shown to increase expression of class II Ag on B cells and induce B cell proliferation. In the murine system, IL-4 induced differentiation of precursor B cells into pre-B cells. In order to analyze growth factors on B cell development we have established an in vitro culture system for human bone marrow cells. We found that in the presence of IL-4, normal human precursor and pre-B cells can be induced to differentiate in the absence of cell proliferation with four days of culture. Furthermore, IL-4 depressed proliferation induced by supernatant from a T cell line. The differentiation was measured by an increase in both the number of cytoplasmic mu and surface IgM-positive cells. The effect of IL-4 on precursor and pre-B cell differentiation was detected as soon as 14 h of exposure to the lymphokine in the absence of an adherent feeder layer. These data suggest that IL-4 directly affects the differentiation process of normal human precursor and pre-B cells, and may antagonistically affect cell proliferation.     

IL-3 and stromal cell-derived factor synergistically stimulate the growth of pre-B cell lines cloned from long-term lymphoid bone marrow cultures

The addition of IL-3 to modified Whitlock-Witte long-term lymphocyte cultures was found to enhance the growth of a small but significant number of B cell precursors supported by an adherent stromal cell monolayer. Several pre-B cell lines were cloned from IL-3-treated long-term lymphocyte cultures. The growth requirements and physical properties of one representative clone, BL/3, are described. BL/3 cells were shown to be unresponsive to IL-3 except when it is used at very high concentrations. In contrast, significant growth was stimulated by stromal cell conditioned medium previously shown to contain a pre-B cell growth factor. Optimal growth of the pre-B cell clone was stimulated by stromal cell conditioned medium plus IL-3. Synergy between the stromal cell-derived factor and IL-3 occurred when IL-3 was used over a wide range of concentrations including a relatively low amount that was ineffective as a growth stimulus by itself. The finding that more than one factor is required to sustain optimal growth of some pre-B cells parallels the complex growth requirements reported for some primitive myeloid/erythroid progenitors.     

Stromal cell regulation of lymphoid and myeloid differentiation

In vitro microenvironmental influences seem to be critical for both B lymphocyte and myeloid differentiation. Studies on murine Dexter cultures and Whitlock-Witte lymphocyte cultures suggest the presence of two critical stromal regulatory cells: an alkaline-phosphatase-positive epithelioid cell and a macrophage. Further data suggest that these cells are capable of producing colony stimulating factor-1, granulocyte-macrophage CSF, a myeloid synergizing activity, and probably separate B cell growth factors. Isolation of a cell line from Dexter stroma was accomplished and this line produced CSF-1, GM-CSF, a pre-B cell and myeloid synergizing activity, and an activity acting on differentiated B cells. We speculate that the Dexter and Whitlock-Witte in vitro culture systems are regulated by factors produced by the two adherent cell types. A lineage nonspecific factor capable of inducing cells into the B lineage or synergizing with interleukin-3, GM-CSF, and CSF-1 is produced, which presumably acts on early stem cells. In addition, the cell line produces GM-CSF, CSF-1, and a factor acting on differentiated B cells. We speculate that in these culture systems, these "terminal differentiating hormones" regulate the final pathway of differentiation, whereas the pre-B-synergizing activity supports early stem cells that can then respond to the other differentiating hormones.     

Interleukin-8 expression in AIDS-associated lymphoma B-cell lines

Interleukin 8 (IL-8), a member of the CXC subfamily of chemokines, is a potent inflammatory cytokine produced by many cell types in response to several stimuli. In an attempt to determine whether human B-cell IL-8 functions as an autocrine growth factor, a wide panel of B-cell lines derived from patients with AIDS-associated B-cell lymphomas (AABCL) (n = 5) and from non-AABCLs (n = 8) was studied for expression of IL-8, IL-8 Receptor type A (IL-8R), and secretion of IL-8 protein. Using RT-PCR and Northern Blot analysis, we were able to observe IL-8 expression ubiquitously. However, IL-8R expression was seen only in EBV negative (4 out of 7) B-cell lines. EBV and HIV-1 activated B-cell line; HBL-1, was the major secretor of IL-8. Our results demonstrate that IL-8 is expressed in malignant B-cell phenotypes that correspond to a narrow window in the B-cell differentiation pathway (pre-B, early-B, and intermediate-B) as well as in normal CD19-enriched B-cells. Furthermore, IL-8 autocrine loops were not evident since IL-8R was detected only in cell lines that did not secrete IL-8 protein.     

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.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

Establishment of a leukemic cell model for studying human pre-B to B cell differentiation

Reproducible models for examining early stages of human B cell differentiation are poorly developed. We now describe the establishment and characterization of a novel human leukemic cell line that recapitulates the pre-B to B cell stage of differentiation. This cell line, designated BLIN-1, was initially established in tissue culture medium containing low m.w. B cell growth factor, and consistently shows a dependency on this cytokine for optimal growth at low density. BLIN-1 cells have a 9p chromosomal abnormality, identical to the abnormality present in the leukemic blasts from the patient's original bone marrow aspirate. The immunologic phenotype of BLIN-1 is consistent with a cell arrested at the pre-B cell stage of development. Analysis of Ig gene rearrangement and Ig expression in a series of BLIN-1 subclones show that the cells spontaneously rearrange kappa light chain genes, leading to the differentiation of surface kappa-negative pre-B cells into surface kappa-positive B cells. The BLIN-1 cell line is, to our knowledge, the first defined human model for examining this critical developmental stage in human B cell ontogeny. As such, it offers a unique resource for examining variables influencing onset of kappa L chain gene rearrangement and expression.     

Bone marrow stromal cell regulation of B-lymphopoiesis. I. The role of macrophages, IL-1, and IL-4 in pre-B cell maturation

Our experiments have addressed regulation of B lymphocyte formation by bone marrow stromal cells. Stromal cells appear to produce a regulatory factor that acts at the pre-B cell stage to induce the expression of Ig L chains and surface Ig. Bone marrow stromal cell conditioned medium was found to contain this factor and the active component was partially purified by HPLC. This stromal cell-derived factor had a m.w. between 16,000 and 20,000, was specifically neutralized by anti-IL-4 mAb, 11B11, and enhanced the proliferation of anti-mu-stimulated B cells. We also found that rIL-4 induced B cell formation in culture. In our studies, IL-1 had no direct effect on pre-B cell maturation, however, IL-1 was found to stimulate the production of IL-4 by both heterogeneous bone marrow stromal cells and a cloned stromal cell line, SCL-160. These effects of IL-1 on factor production by stromal cells were duplicated by the addition of bone marrow-derived macrophages to SCL-160 cells. We conclude that stromal cell-derived IL-4 is a physiologic stimulator for B cell generation. In addition, macrophages appear to play a role in B cell formation by regulating the production of IL-4 by stromal cells via the secretion of IL-1.     

Induction of murine lymphokine-activated killer cells by recombinant IL-7

The data demonstrate that IL-7, a cytokine that was originally identified, purified, and cloned based upon its ability to support the growth of pre-B cells in vitro, also induces proliferation and promotes the generation of lymphokine-activated killer (LAK) cell activity in populations of resting peripheral lymphoid cells. Although the kinetics of LAK induction by IL-7 (which peaked at days 6 to 8 of culture) was slower than that detected in cultures containing IL-2 (which peaked at day 4), IL-7 was significantly more effective at maintaining cytotoxic activity over longer periods of time, and greater viable cell recoveries, than was IL-2. A wide range of murine tumor target cells were found to be lysed in an MHC-unrestricted fashion by IL-7 induced LAK, but syngeneic Con A-induced lymphoblasts were not; nor were target cells from the human tumors K562 or Daudi lysed by IL-7 LAK. IL-7 LAK were induced in populations of lymphoid cells obtained from secondary lymphoid tissues (peripheral lymph nodes and spleen), but not from primary lymphoid tissues (thymus and bone marrow). LAK induced by IL-7 from unfractionated populations of lymphoid cells were completely eliminated by treatment with anti-CD8 or anti-Thy-1+C, and unaffected by treatment with anti-CD4, anti-asialo GM1 or anti-NK1.1+C. Interestingly, although no detectable CD4+ effector cells could be detected in populations of LAK generated from unfractionated populations of lymphoid cells stimulated by IL-7, they were found to be generated from populations of lymphoid cells from which CD8+ cells had been eliminated before being cultured in medium containing IL-7. These data suggest that CD4+ T cells do not normally give rise to IL-7-induced LAK unless they are first separated from CD8+ T cells. LAK induced by IL-7 appear to be distinct from LAK activity induced by IL-2 in that there is no detectable involvement of NK-like effector cells at either the precursor or effector cell stages.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.