Long-term Bone Marrow Culture System Research Articles

Novel three-dimensional long-term bone marrow culture system using polymer particles with grafted epoxy-polymer-chains supports the proliferation and differentiation of hematopoietic stem cells

Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. We examined the hematopoietic supportive ability of stromal cells in a 3D culture system using polymer particles with grafted epoxy polymer chains. Umbilical cord blood-derived CD34(+) cells were co-cultivated with MS-5 stromal cells. They formed a 3D structure in the culture dish in the presence of particles, and the total numbers of cells and the numbers of hematopoietic progenitor cells, including colony-forming unit (CFU)-Mix, CFU-granulocyte-macrophage, CFU-megakaryocyte and burst-forming unit-erythroid, were measured every seven days. The hematopoietic supportive activity of the 3D culture containing polymer particles and stromal cells was superior to that of 2D culture, and allowed the expansion and maintenance of hematopoietic progenitor cells for more than 12 weeks. Various types of hematopoietic cells, including granulocytes, macrophages and megakaryocytes at different maturation stages, appeared in the 3D culture, suggesting that the CD34(+) cells were able to differentiate into a range of blood cell types. Morphological examination showed that MS-5 stromal cells grew on the surface of the particles and bridged the gaps between them to form a 3D structure. Hematopoietic cells slipped into the 3D layer and proliferated within it, relying on the presence of the MS-5 cells. These results suggest that this 3D culture system using polymer particles reproduced the hematopoietic phenomenon in vitro, and might thus provide a new tool for investigating hematopoietic stem cell-stromal cell interactions.

A probable role for trail-induced apoptosis in the pathogenesis of marrow failure: Implications from an in vitro model and from marrow of aplastic anemia patients

Aplastic anemia (AA) is a syndrome of hematopoietic failure involving increased apoptosis of stem cells. In order to investigate the molecular mechanisms participated in the process of marrow failure, we created an in vitro model of hematopoietic cell suppression, by continuous addition of TNF-α and IFN-γ in an vitro long-term bone marrow culture system. An up-regulation of Fas expression was observed in CD34 + cells in cytokine treated cultures, compared to controls. This was accompanied by significant TRAIL and decreased caspase 3 mRNA expression, whereas the expression of Bcl-2 family members was low (Bcl-xl) or absent (Bcl-2, Bax). The expression of these apoptotic genes was also investigated in aplastic anemia patients. Apart from Fas mRNA expression in total marrow and/or CD34 + cells, TRAIL mRNA expression was found only in CD34 + cells in active disease while in total marrow cell compartment this remains a constant finding even in patients in remission. The above data are in agreement with previous studies proposing a major role for the extrinsic apoptosis pathway in the pathogenesis of aplastic anemia and additionally introduce TRAIL as a probable important molecule in the process.

Role of VLA-4 and VLA-5 in ex vivo maintenance of human and pig hematopoiesis in human stroma-supported long-term cultures

The advantage of recipient hematopoiesis over that of xenogeneic donors poses a fundamental obstacle to the induction of xenograft tolerance through mixed hematopoietic chimerism. Here we explore the role of beta1 integrins in maintenance of human vs porcine hematopoiesis within a human hematopoietic environment. Porcine and human c-kit+ bone marrow cells were purified and cultured on human bone marrow stroma for 6 weeks. The role of VLA-4 and VLA-5 in the maintenance of porcine vs human hematopoiesis in this human stroma-supported long-term bone marrow culture (LTBMC) system was evaluated by using blocking mAbs that bind to both species. Blocking VLA-4 with HP2/1 inhibited both human and porcine hematopoiesis, whereas anti-VLA-5 (SAM-1) suppressed the function of human, but not porcine, hematopoietic cells. In mixed LTBMC of porcine and human cells on a human stroma, porcine hematopoietic cells were at a competitive disadvantage, as seen by a rapid decline in cellularity, including clonogenic progenitors. This disadvantage was substantially overcome by the addition of SAM-1. Furthermore, human, but not porcine, cell adhesion to human fibronectin was inhibited by arginine-glycine-aspartic acid (RGD) peptides. Taken together, these results indicate that VLA-4 plays critical role for porcine hematopoiesis in a human hematopoietic environment, and raise the possibility that porcine VLA-5 might be unable to bind the respective human ligand and/or to initiate adequate post-ligand-binding signaling. Thus, VLA-5 may provide a potential target for developing approaches to improve porcine hematopoiesis in human recipients.

Evaluation of mmp and timp expression in the hematopoietic microenvironment

It is now recognized that the extracellular matrix (ECM) of bone marrow (BM) plays a role not only in supporting BM structure but also in regulating cellular functions. Matrix metalloproteinases (MMPs) and their nature tissue inhibitors (TIMPs) regulate ECM turnover and TIMPs also regulate cell growth. As human long-term bone marrow culture (LTMC) mimics the hematopoietic microenvironment, we used this model to examine the secretion of MMPs and TIMPs. To evaluate the contribution of individual lineages we examined the expression of MMP-2, MMP-9, MT-1 MMP, MT-4 MMP and TIMPs-1, -2 and -3 by hematopoietic cells of erythroid, myeloid and megakaryocytic lineages (expanded in vitro from human normal BM CD34+ cells) and by BM stromal cells (CFU-F, endothelial cells). We found that MMP-9 is expressed and secreted by precursors of all hematopoietic lineages while MMP-2 and MT-1 MMP are expressed mainly by stromal cells and cells of myeloid and megakaryocytic but not erythroid lineages. BM stromal cells expressed and secreted high levels of all the TIMPs tested. This pattern of secretion of MMPs and TIMPs was reflected in the LTMC system: during the first week of LTMC MMP-9 levels were high and then decreased with reduced number of hematopoietic cells while levels of MMP-2, TIMP-1 and TIMP-2 increased with establishment of an adherent layer and expansion of stromal cells. TIMP-3 was released from the ECM matrix by the end of week 8–9 when the production of CFU-GM by adherent and nonadherent layers decreased significantly. Thus we present clear evidence that MMPs and TIMPs are constituents of the hematopoietic microenvironment and we are now investigating their role in hematopoiesis.

CROSS-SPECIES COMPATIBILITY OF INTERCELLULAR ADHESION MOLECULE-1 (CD54) WITH ITS LIGANDS1

The molecular interactions of intercellular adhesion molecule-1 (ICAM-1; CD54) are potentially important in several situations in the context of pig-to-human xenotransplantation. If porcine bone marrow is to be used for the induction of xenograft tolerance in humans, the role that has been suggested for ICAM-1 in the interactions of haematopoietic stem cells makes its cross-species compatibility important. Similarly, the potential role of ICAM-1 interactions in graft rejection makes it an important molecule to study. An in vitro static cell-to-cell adhesion study was used to look at the successful interaction of ICAM-1 with its ligands across the pig-human species barrier in both directions. A second in vitro system, the standard long-term bone marrow culture (LT-BMC), was used to study the functional role of ICAM-1 in haematopoiesis. Human ICAM-1 was able to adhere to ligands on porcine cells, including one or more ligand that contains CD18. Conversely, human CD18-containing ligands mediated adherence to porcine cells. Using the long-term bone marrow culture system, there was no evidence that blocking the interactions of ICAM-1 inhibited hematopoiesis, either in the human-human or pig-human combinations of precursor cells and marrow stroma. ICAM-1 is able to interact with at least some of its ligands across the species barrier, in both pig-human and human-pig combinations. However, the interactions of ICAM-1 do not appear to be central to hematopoiesis, at least in the model system used.

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5–10 days of culture, but declined to low levels at 15–20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34+cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

In VivoDisturbance of Hematopoiesis in Mice Persistently Infected with Murine Cytomegalovirus: Impairment of Stromal Cell Function

Although the pathogenic effects of a primary cytomegalovirus (CMV) infection on hematopoiesis has been largely investigated so far, the effects of a persistent or latent infection have yet to be elucidated. The effects of persistent CMV infection on hematopoiesis thus were examined using BALB/c mice at 4 weeks postinfection with 0.2 LD50of murine CMV (MCMV) infection as a persistent infection model. The parameters of constitutive hematopoiesis of MCMV persistently infected mice were completely identical to those of the control. However, the inductive hematopoiesis, examined by the autologous marrow reconstitution after 5-fluorouracil administration, was significantly impaired in the MCMV persistently infected mice (P< 0.05). In a colony-forming unit-spleen assay and a long-term bone marrow culture system, a decreased capacity of bone marrow stromal cells to support hematopoiesis was observed in the MCMV-infected mice in comparison with the controls. The existence of MCMV DNA in the adherent cells of long-term bone marrow culture from the MCMV-infected mice were confirmed by a polymerase chain reaction but not in the nonadherent cells. Furthermore, the increased expression level of tumor necrosis factor-α by stromal cells was also observed by semiquantitative reverse transcriptase-polymerase chain reaction. These results therefore strongly suggest that MCMV remains to infect the stromal cells while also inhibiting inductive hematopoiesis through the impairment of the stromal cell functions in the MCMV persistently infected mice.

Stimulatory effects of neopterin on hematopoiesis in vitro are mediated by activation of stromal cell function.

The pteridine neopterin (NP) was shown to be produced by monocytes and is known to be a useful marker of immunological activation, although, its biological activity is still unclear. Recently, we found that intravenous administration of NP increased the numbers of blood leukocytes, and granulocyte-macrophage progenitor cells (CFU-GM) in the bone marrow and spleens of mice. In order to elucidate the mechanism whereby NP stimulates hematopoiesis, the effects of NP on hematopoietic stem cell proliferation and differentiation in vitro were studied using a long-term bone marrow culture (LTMC) system with cloned stromal cell line, MS-5. Adding NP to the LTMC increased the numbers of cells in total, CFU-GM and colony-forming unit in spleen (CFU-S). NP also increased the number of CFU-GM in a soft agar culture system, but it did not enhance CFU-GM colony formation when target bone marrow cells were semi-purified (T, B and adherent cell-depleted bone marrow cells) and cultured in this system, suggesting that NP did not directly affect the proliferation of hematopoietic progenitors. Conditioned medium obtained from NP-treated stromal cells had much greater colony-stimulating activity than that obtained from untreated stromal cells. Furthermore, NP treatment stimulated the production of IL-6 and GM-CSF by stromal cells. All these findings suggest that NP stimulates hematopoietic cell proliferation and differentiation in vitro by activating stromal cell function.

Identification of an IL-7-Associated Pre-Pro-B Cell Growth-Stimulating Factor (PPBSF). II. PPBSF Is a Covalently Linked Heterodimer of IL-7 and aMr 30,000 Cofactor

AbstractEvidence is provided in a companion paper for an IL-7-associated molecular complex that selectively stimulates the proliferation and presumptive differentiation of pre-pro-B cells in our long-term bone marrow culture system and “primes” them to proliferate in response to monomeric IL-7. Here, Western immunoblot analysis reveals that this pre-pro-B cell growth-stimulating factor (PPBSF) is a self-assembling heterodimer of IL-7 and a cofactor with a Mr of 30,000. Thus, when developed with anti-IL-7 mAb, PPBSF migrates electrophoretically as a covalently bound ∼55-kDa molecule under nonreducing conditions but dissociates under reducing conditions. Furthermore, the addition of rIL-7 or native IL-7 to medium conditioned by stromal cells from IL-7 gene-deleted (−/−) mice results in the formation of active 45-kDa and 55-kDa molecular complexes, respectively. Antiserum prepared in IL-7(−/−) mice against affinity-purified PPBSF contained separable reactivities for IL-7 and the non-IL-7 component of PPBSF. The PPBSF cofactor detected by this antiserum migrates as an ∼30-kDa molecule and is able to maintain the viability, but not the proliferation, of pre-pro-B cells. Furthermore, the cofactor is produced constitutively by IL-7(−/−) and IL-7(+/+) bone marrow stromal cells under pro-B- but not pre-B-type culture conditions. Consequently, IL-7 appears to exist almost entirely as a heterodimer (i.e., PPBSF) in pro-B-type cultures, whereas it exists almost entirely as a monomer in pre-B-type cultures. Although the identity of the PPBSF cofactor remains to be determined, it does not appear to be stem cell factor, insulin-like growth factor-1, thymic stromal-derived lymphopoietin, flt3, stromal cell-derived factor-1, or IL-7R.

ONCOSTATIN M STIMULATES MACROPHAGE-POLYKARYON FORMATION IN LONG-TERM HUMAN BONE-MARROW CULTURES

Though oncostatin M (OSM) is a potent mediator of the inflammatory reaction, its role in inflammation and bone resorption is still unclear. A long-term bone-marrow culture system is usually developed to allow the formation of multinucleated cells (MNC) and was used here to define the effects of human recombinant OSM on human MNC formation. OSM significantly upregulated (1.9- to 5.6-fold) the number of MNC in these cultures in a dose- and time-dependent manner. Cell nucleation and tartrate-resistant acid phosphatase activity were also increased. MNC did not display osteoclast characteristics, such as response to calcitonin and failure to resorb dentin surface. However, they expressed a non-specific α-naphthyl acetate esterase as well as macrophage differentiation antigens (CD11b, CD13 and CD33) and were able to perform phagocytosis. Similar effects were observed after addition of 1α,25-dihydroxyvitamin D3. Moreover, in these culture conditions, human bone-marrow mononuclear cells were capable of low-grade resportion in the presence of bone-marrow stromal cells. This low grade resorption was significantly inhibited by addition of 25 ng/ml OSM. Our data demonstrate for the first time that human recombinant OSM significantly stimulates the formation of MNC and could be involved in the inflammatory process via macrophage-polykaryon formation from human bone marrow.

Loss of retroviral gene expression in bone marrow reconstituted mice correlates with down-regulation of gene expression in long-term culture initiating cells.

The question of whether a retroviral vector is expressed in vivo after infection of hematopoietic stem cells remains unpredictable. In this study, we show that a switch off of retroviral sequences can be tested in vitro using the long-term bone marrow culture system. The time kinetics of the down-regulation of retroviral expression in vitro and in vivo have been demonstrated to be similar. The correlation of switch off events in both systems allows for evaluation of retroviral expression in vitro provided the silencing is caused by viral sequences. This approach could be useful for testing vectors for gene therapeutical applications of the hematopoietic system.

Development of B cells in aged mice: decline in the ability of pro-B cells to respond to IL-7 but not to other growth factors.

The process of B lymphopoiesis changes with increasing age, resulting in a severe drop in the number of pre-B cells in old mice. We have shown that the ability of freshly isolated pro-B cells to proliferate on stromal cells declines with age. In this study, we wanted to determine the reason for the diminished response of the aged pro-B cells. The functional alterations could arise from changes intrinsic to the pro-B cells or to composition of the precursor population. Changes in the composition of pro-B cells in marrow or long-term bone marrow culture system for B lymphocytes (LTBMC-B) could not account for the functional losses. We then examined the hypothesis that the diminished amount of proliferation on stromal cells was due to changes in the responsiveness to stroma-derived cytokines. The proliferation to IL-7 but not to stem cell factor (SCF) or insulin-like growth factor 1 (IGF-1) was severely impaired by 24 mo of age, independently of the concentration of IL-7 and length of culture time. The reduced IL-7 response could not be explained by an increase in the percentage of cells undergoing apoptosis; rather, a greater frequency of aged cells remained in G0/G1 after IL-7 stimulation. Furthermore, the impaired responsiveness of the aged pro-B cells is not due to diminished expression of the IL-7R alpha-chain or the common gamma-chain. Since only IL-7 responsiveness is impaired, we believe that the underlying molecular mechanism for the cellular alterations is specific to signaling through the IL-7 receptor complex.

Possible involvement of bone marrow stromal cells in agranulocytosis caused by vesnarinone treatment.

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

Long-term bone marrow cultures in aplastic anaemia.

The long-term bone marrow culture (LTBMC) system provides an in vitro physiological model for the study of stromal cell mediated haemopoiesis in patients with aplastic anaemia. The two aspects of haemopoiesis--stromal and stem cell function--can be analysed separately using a modification of LTBMC with cross-over studies. Patients with aplastic anaemia universally demonstrate defective stem cell function in terms of reduced or absent marrow repopulating ability, reflecting a deficiency of long-term culture initiating cells. Defects in stromal cell function, as assessed by the ability of aplastic anaemia stroma to support normal generation of haemopoietic progenitors, are not common, but may conceal an isolated deficiency of a particular growth factor in some patients due to the overlapping nature of haemopoietic growth factor activities. The stem cell abnormality in aplastic anaemia reflects a deficiency in cell numbers, as well as dysfunction in certain cases. An increased level of apoptosis in aplastic anaemia marrow CD34+ cells exists, and this correlates well with disease severity. LTBMC studies demonstrate that more of the haemopoietic cells are nonviable (apoptotic and dead) compared with normal controls, and this correlates with reduced colony (CFU-GM) generation. An increase in apoptosis among primitive haemopoietic cells may contribute to the stem cell defect in aplastic anaemia. Haemopoietic growth factors such as G-CSF, when given after immunosuppressive therapy such as antilymphocyte globulin and cyclosporin for aplastic anaemia, may act partly by reducing the increased level of apoptosis, resulting in improved stem cell survival.

Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells.

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (+/- 15) and 31 (+/- 21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37 degrees C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Autologous Activated Natural Killer Cells Suppress Primitive Chronic Myelogenous Leukemia Progenitors in Long-Term Culture

A graft-versus-leukemia effect has been well documented to prevent relapse in chronic myelogenous leukemia (CML) after allogeneic marrow transplantation. One type of lymphocytes that may contribute to this effect are natural killer cells (NK), which after activation with interleukin (IL)-2, exhibit a broad range of cytolytic activity against allogeneic and autologous cells. We have previously demonstrated that IL-2-activated NK (ANK) can be generated from blood of patients with CML and are benign in origin. Their proliferation and function, however, diminish with disease progression in CML, suggesting a role in tumor surveillance. We studied the effect of IL-2-activated NK (ANK) on normal and malignant primitive and committed progenitors in a novel long-term bone marrow culture (LTBMC) assay. Because ANK destroy marrow stromal layers, the use of classic stroma-dependent long-term cultures is not possible. Therefore, we used the stroma noncontact LTBMC system developed in our laboratory to analyze the effect of autologous ANK cells on primitive hematopoietic progenitors. Autologous ANK (CD56+/CD3-) were generated from the peripheral blood of 10 patients with chronic phase CML and from six normal individuals by culturing CD5/CD8-depleted mononuclear cells for 14 days in 1,000 U/mL IL-2. At the same time ANK cultures were initiated, sorted normal (CD34+/DR+) marrow populations were plated in Transwell inserts of the stroma noncontact culture. On day 15, hydrocortisone, which rapidly inhibits ANK function, was removed, and autologous ANK were added to the Transwell inserts with fresh LTBMC medium without hydrocortisone but supplemented with 1,000 U/mL IL-2. After 48 hours, the number of colony-forming cells (CFC) was enumerated in methylcellulose culture. To determine the effect of ANK on more primitive long-term culture-initiating cells (LTCIC), the IL-2-supplemented LTBMC medium was replaced with fresh hydrocortisone containing LTBMC medium, and cultures were maintained for an additional 5 weeks. We demonstrate that autologous ANK did not suppress normal CFC or LTCIC. In contrast, ANK from eight patients with CML with potent cytotoxicity against NK-sensitive (K562) NK-resistant (Raji) tumor targets exhibited an ANK dose-dependent suppression of both CFC and LTCIC. Interestingly, ANK from two patients with CML who exhibited diminished cytotoxicity also did not suppress autologous CFC and LTCIC. These studies indicate that ANK with potent major histocompatibility complex unrestricted cytotoxic activity suppress malignant hematopoiesis. This effect was not mediated by soluble factors and was absolutely dependent on direct cell-to-cell contact. We further demonstrate that the beta2 integrin receptor is involved in ANK recognition of CML targets. These observations support the use of autologous ANK therapy to prevent relapse of CML after autologous marrow transplantation or use of ANK to purge CML marrow for autologous transplantation.

TNF-alpha inhibits the further development of committed progenitors while stimulating multipotential progenitors in mouse long-term bone marrow cultures.

Abstract We studied simultaneously the effect of TNF on both relatively primitive and more mature hematopoietic progenitors by using a modification of the mouse long-term bone marrow culture system of Dexter. Mycophenolic acid-treated long-term bone marrow cultures were charged with stromally depleted bone marrow cells along with 10 to 500 U/ml recombinant human TNF-alpha, and assayed each week for 5 wk for the presence of primitive (CFU-S and high proliferative potential colony-forming cell) and mature (CFU-C) colony-forming units. The results demonstrate that TNF-alpha has differential effects on hematopoiesis; it virtually eliminates CFU-C, significantly lowers high proliferative potential colony-forming cell (CFC) numbers and increases CFU-S numbers. This effect appears to be independent of CSF production in the cultures as shown by protein assays. Northern blot analyses showed no differences in expression of CSF genes or any expression of those encoding for IL-1, IL-3, or IL-6. These results, obtained in a long-term culture system that closely mimics in vivo bone marrow, demonstrate that many of the apparently contradictory effects of TNF observed in vitro occur concomitantly and are attributable to the presence of cells at various stages of maturity in the cultures. The data suggest a mechanism of this activity in which TNF is able to block the differentiation of primitive progenitors even as they are stimulated to proliferate.

Role of CD44 in the development of natural killer cells from precursors in long-term cultures of mouse bone marrow.

The role of the adhesion molecule CD44 in the development of NK cells was analyzed in a mouse long-term bone marrow culture system. After 4 wk of culture (day 0), recombinant human IL-2 was added and 13 days later the cells generated were shown to have substantial cytotoxic activity against YAC-1 and to be enriched for NK cells, as assessed for NK-1.1 phenotype by flow cytometric analysis. Physical separation between stroma and precursors partially inhibited proliferation and, consequently, a lower number of cytotoxic cells were produced. Similar results were obtained when an anti-CD44 mAb was added together with IL-2 at day 0. The disruption of hyaluronic acid (HA), one of the ligands of CD44, by hyaluronidase or the competition for the binding of CD44 by soluble HA added with IL-2 on day 0 inhibited both proliferation and development of cytotoxicity to a greater degree than did anti-CD44. These results indicate that interaction of CD44 with HA plays an important role in the development of pre-NK cells into cytotoxic effector cells.

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.