The TLR1/2 agonist PAM(3)CSK(4) instructs commitment of human hematopoietic stem cells to a myeloid cell fate.

Toll-like receptors (TLRs) constitute a family of nonpolymorphic receptors that are devoted to pathogen recognition. In this work, we have explored the impact of TLR ligands (TLR-L) on human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We show that HSCs and HPCs have a comparable pattern of expression of TLR transcripts characterized by the predominance of TLR1, -2, -3, -4 and -6. In long-term cultures of HSCs, HPCs and stromal cells, most TLR-L profoundly inhibited B-cell development while preserving or enhancing the production of myeloid cells. In short-term cultures, the TLR1/2 ligand PAM(3)CSK(4) induced a large proportion of HPCs to express markers of the myelomonocytic lineage. PAM(3)CSK(4) induced only marginal expression of myeloid lineage markers on HSCs but promoted their myeloid commitment as revealed by their acquisition of the phenotype of multi- and bipotential myeloid progenitors and by upregulation of the transcription factors PU.1, C/EBPalpha and GATA-1. Our results suggest that TLR agonists can bias the lineage commitment of human HSCs and shift the differentiation of lineage-committed progenitors to favor myelopoiesis at the expense of lymphoid B-cell development.

CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells.

Human CD34+ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor-receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.

Heterogeneity of the inhibitory effects of IL-4 in two novel B lineage acute lymphoblastic leukemia cell lines.

The present study describes two novel cell lines, DUNATIS and SILVANUS, established from B lineage acute lymphoblastic leukemia patients. Respectively, DUNATIS and SILVANUS display an early pre-B cell and a pre-B cell phenotype. Spontaneous DNA replication of both cell lines was strongly inhibited by IL-4. This effect was directly mediated by IL-4 and exerted through the CD124 IL-4 receptor chain. Notably, IL-4 was associated with rapid cell death and reduction of cellularity in DUNATIS, whereas these parameters were considerably less pronounced and only observed after longer-term exposure of the SILVANUS cells to IL-4. In addition to these differences, although both cell lines expressed FES oncoprotein, a 100 kDa protein associated with FES was strikingly found to be tyrosine-phosphorylated in response to IL-4 exclusively in DUNATIS cells. These data demonstrate that IL-4 displays heterogenous effects on leukemic B cell precursors responsive to inhibition of DNA synthesis via IL-4 mediated engagement of the CD124 receptor chain. The present findings may be of use for appreciation of the effects of IL-4 in B lineage ALL, and the novel cell lines could represent a model for further identification of target molecules in IL-4 signalling.

Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand.

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti-CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.

Distribution of carboxypeptidase M on lymphoid and myeloid cells parallels the other zinc-dependent proteases CD10 and CD13.

Monoclonal antibody (MoAb) M27 was generated after immunization of mice with the human B-lineage acute lymphoblastic leukemia cell line Pre-ALP. Under reducing conditions, MoAb M27 precipitated a 60-kD surface-membrane molecule from Pre-ALP cells. Expression cloning of Pre-ALP cDNA showed that M27 recognizes carboxypeptidase M (CPM), a cell-surface, zinc-dependent protease known to cleave off basic C-terminal amino acids from peptide hormones. Using M27 antibody, CPM was detected only at discrete B lymphocyte developmental stages, namely on committed precursors and on germinal center cells. CPM was also expressed on mature T cells, mainly after activation. These results provide the first description of a carboxy-peptidase on lymphoid cells. In addition, CPM was found on granulocytes and monocytes, but not on their progenitors. Strikingly, CPM was present only on CD38+ cells, irrespective of lineage affiliation. Of interest, CPM displayed a largely overlapping distribution with the CD10 and CD13 peptidases, with which it shares common substrates (enkephalins, bradykinin). Collectively, the present data show a previously unrecognized distribution pattern of CPM on lymphoid and myeloid cells and suggest that CPM may cooperate with CD10 and CD13 to regulate biologic activity of peptide hormones on leukocytes.

Proliferation of MIELIKI a novel t(7;9) early pre-B acute lymphoblastic leukemia cell line is inhibited concomitantly by IL-4 and IL-7.

The present study describes a novel cell line, MIELIKI, established from bone marrow of a pediatric patient with B lineage acute lymphoblastic leukemia (ALL) at diagnosis. The MIELIKI cell line displays an early pre-B cell phenotype (CD10+, CD19+, CD20+, CD34-, Cmu-, sIg-) with rearrangements on both Ig heavy chain and k light chain alleles, and carries an unfrequent t(7;9) chromosomal translocation identical to the freshly isolated leukemic blasts. The proliferation of MIELIKI cells was abrogated by IL-4 and by IL-7, as measured by DNA replication and viable cell recovery. The effects of IL-4 and IL-7 were mediated, respectively, through the CDw124 and CDw127 IL-4 and IL-7 receptor components. Growth inhibition by IL-4 was not mediated by soluble factors released by MIELIKI cells in response to IL-4, suggesting the existence of an intrinsic negative signaling pathway. Finally, neither IL-4 nor IL-7 were found to induce maturation of MIELIKI into cells expressing cytoplasmic or surface membrane mu chain. The present cell line should constitute a useful model of t(7;9) early pre-B ALL and allow investigation of the relationship between IL-4 and IL-7 negative signaling in leukemic B cell ontogeny.

A surrogate 15 kDa JC kappa protein is expressed in combination with mu heavy chain by human B cell precursors.

A novel kappa protein, encoded by a germline JC kappa transcript, is expressed by normal and leukemic human B cell precursors. The transcript displays an open reading frame initiated by a non-AUG codon, and predicts a 15 kDa molecule which could be readily confirmed by in vitro translation. Cellular expression was demonstrated by immunofluorescence, precipitation and Western blotting. Furthermore, 2-D gel electrophoresis revealed that germline JC kappa can covalently associate with mu heavy chain at the surface of pre-B cells. We therefore propose that during B cell lymphopoiesis, two alternative pathways could be operative in which mu heavy chain can either associate with lambda 5 or germ-line JC kappa.

Interleukin-13 inhibits the proliferation of normal and leukemic human B-cell precursors.

Interleukin-13 (IL-13) is a T-cell-derived cytokine that displays homology with IL-4 and shares some of its biologic functions. We investigated the effects of IL-13 on normal human B-cell precursors (BCP) and their malignant counterparts in B-lineage acute lymphoblastic leukemia (BCP-ALL). IL-13 inhibited growth of CD19+ slg- normal BCP cultured in the presence or absence of bone marrow accessory stromal cells and IL-7. In addition, IL-13 inhibited proliferation of blasts isolated from leukemic patients and cells from established BCP-ALL lines. Differences were observed in a number of cases with respect to growth inhibition in response to IL-13 and IL-4. These results suggest heterogeneity in the expression of IL-13 and IL-4 receptors in B-cell ontogeny. Growth-inhibition by IL-13 could be reverted by anti-IL-4 receptor antibody, indicating that the IL-13 and IL-4 binding chains can be closely associated on BCP. We further showed that the inhibitory effect of IL-13 results from decreased cell-cycle activity. Finally, whereas IL-13 induced CD23 expression on BCP-ALL cells, it did not promote differentiation into slg+ B lymphocytes.

Activated CD4+ T cells induce CD40-dependent proliferation of human B cell precursors.

Anti-CD3-activated human CD4+ T cell clones were found to induce proliferation of CD10+, CD19+, surface(s) Ig- B cell precursors (BCP) isolated from human fetal bone marrow. The great majority of the B lineage cells recovered in cocultures of BCP and activated T cells displayed a BCP phenotype (Ig- or cytoplasmic mu+ and kappa/lambda-), including most of the cycling cells, indicating that the cultures do not favor a transition to mature B cells. Supernatants of activated T cells were ineffective in inducing BCP proliferation, indicating the necessity of close association with stimulator cells. In line with this finding, the CD40 molecule was found to represent an important component of the cocultures, as BCP proliferation was strongly inhibited by soluble anti-CD40 antibody. In addition, CD4+ T cell clones from a hyper-IgM patient expressing a truncated CD40 ligand (CD40-L) failed to induce BCP proliferation. Finally, a combination of cytokines (IL-2, IL-3, IL-7, and IL-10) enhanced the observed T cell-dependent BCP proliferation, but could not substitute for the deficient CD40-L. Taken together, our data demonstrate that CD4+ T cells exert a stimulatory effect on in vitro B human lymphopoiesis via the CD40 pathway. The present results suggest that T cells may play an important role in regulating B cell ontogeny in the bone marrow.

Myofibroblastic stromal cells isolated from human bone marrow induce the proliferation of both early myeloid and B-lymphoid cells.

Normal human bone marrow stromal cells (BMSC) were isolated from Dexter-type long-term cultures according to their capacity to adhere to plastic and to their lack of hematopoietic antigens. The BMSC displayed a homogeneous appearance and a myofibroblastic phenotype in culture. The stromal cells (SC) were shown to support the proliferation of purified CD34+ hematopoietic progenitors and permitted us to maintain myeloid cells for several weeks in culture. In addition, the BMSC induced the proliferation of purified CD10+ s mu- fetal BM B-cell precursors (BCP). The capacity of the BMSC to induce the proliferation of early myeloid cells was shared by several other human fibroblastic-like cell types. In contrast, the BMSC were far superior to other adherent cells for induction of BCP proliferation. This capacity was largely mediated by endogenously produced interleukin-7 (IL-7), because it could be inhibited by anti-IL-7 antibody. In line with this finding, addition of IL-7 considerably enhanced BCP proliferation in cocultures with skin fibroblasts or synoviocytes. Thus, production of IL-7 appears to be a critical parameter that determines the ability of fibroblastic-like cells to induce BCP proliferation. Taken together, our data show that normal human myofibroblastic BMSC induce the proliferation of both early myeloid and B-lymphoid cells in the absence of accessory hematopoietic cells. The present system should constitute a model to study interactions between native human BM myofibroblastic stroma and various hematopoietic cell subsets.

Human B cell precursors proliferate and express CD23 after CD40 ligation.

The CD40 surface membrane molecule plays an important role in the activation of mature human B cells, but its role in earlier stages of B lineage development is unknown. Here, we have investigated the effects of triggering the CD40 antigen on B cell precursors (BCP) by crosslinking with anti-CD40 antibody presented by Fc gamma-receptor type II-transfected murine Ltk- cells (CD40 system). CD10+ surface immunoglobulin negative (sIg-) BCP, freshly isolated from fetal bone marrow or precultured on stromal cells, proliferated in the CD40 system. This effect required the presence of IL-3, which acted as a specific cosignal among a panel of cytokines examined. The association of IL-10 and IL-7 potentiated the observed IL-3 and CD40-dependent BCP proliferation, demonstrating that IL-10 can act on early B lineage cells. CD40-dependent activation of fetal BCP did not favor maturation to sIg+ B cells, but resulted in the induction of high levels of surface membrane CD23. The emerging CD23+ BCP lacked sIg and CD10, and represented an important proportion of the cycling cells in the CD40-dependent cultures. Taken together, our data demonstrate that stimulation of the CD40 antigen induces expression of the CD23 gene, and regulates cell proliferation during normal human B cell ontogeny.

Tumor necrosis factor alpha cooperates with interleukin 3 in the recruitment of a primitive subset of human CD34+ progenitors.

We have recently demonstrated that tumor necrosis factor alpha (TNF-alpha) potentiates interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor-induced growth of CD34+ hematopoietic progenitor cells (HPC), and favors the generation of dendritic/Langerhans cells. The stimulatory effect of TNF-alpha was detailed in the present study. Thus, CD34+ HPC entering in cycle (S/G2M) after a 48-h pulse with IL-3 expressed the transferrin receptor (TfR), and fluorescence-activated cell sorter-separated TfR+ HPC, but not TfR-HPC, showed a high proliferative response to IL-3. In contrast, TfR-HPC were found to undergo strong proliferation in response to IL-3 + TNF-alpha. Limiting dilution experiments indicated that TNF-alpha increased both the frequency and the average size of clones generated from TfR-HPC as a result of the development of a higher number of large clones. In contrast, TNF-alpha did not enhance the IL-3-dependent proliferation of TfR+ HPC. Preculturing CD34+ HPC for 48 h with TNF-alpha enhanced the subsequent generation of IL-3-dependent colony-forming units. Precultures with TNF-alpha or cultures with suboptimal doses of TNF-alpha allowed the recruitment of cells with both granulocytic and monocytic differentiation potential. Taken together, our results indicate that TNF-alpha recruits a subpopulation of CD34+ HPC hyposensitive to IL-3, with high proliferative capacity and some features of multipotential progenitors, that are likely to be more primitive than those responding to IL-3 alone.

Culture of human fetal B-cell precursors on bone marrow stroma maintains highly proliferative CD20dim cells.

Growth of human B-cell precursors (BCP) was achieved by plating fetal CD10+ surface-mu (s mu)- cells in liquid medium onto bone marrow-derived fibroblastic stromal cell layers deprived of hematopoietic cells. Proliferation of the fetal BCP was strongly potentiated by the addition of interleukin-7 (IL-7) to the cultures. Cultures included both a stroma-adherent and -nonadherent fraction of lymphoid cells, allowing us to expand the number of input BCP to 13-fold. In the presence of exogenous IL-7, proliferation was dose-dependent relative to the number of stromal cells, demonstrating that soluble IL-7 does not act alone to promote optimal growth. We further showed that the lymphoid cells recovered remain CD10+ sIg- BCP and that most cells expressed the maturation-associated CD20 antigen when IL-7 was added to the cultures. Whereas both freshly isolated CD20- and CD20bright BCP proliferated in the presence of stroma, we observed that high-proliferative capacity CD20dim cells were maintained in the cultures. Finally, CD20dim sorted cells were shown to subsequently acquire high levels of CD20 expression in culture, thus demonstrating a partial maturation sequence. The present culture system thus represents a useful model for studying the regulatory signals in early human B lymphopoiesis.

Interleukin 4 inhibits in vitro proliferation of leukemic and normal human B cell precursors.

In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells.

Growth and maturation requirements of human B cell precursors.

The present study describes our efforts to induce the proliferation of human B cell precursors (BCP). Committed BCP (CD10+, sIgM-) isolated from fetal bone marrow (18-25 weeks) were induced to proliferate at low levels in the presence of IL7. IL3 potentiated this effect of IL7 on BCP, while IL4 partially inhibited this proliferation. However, neither of these cytokines allowed the emergence of mature B cells. The growth of BCP was strongly potentiated by the presence of an adherent fibroblastic bone marrow stromal layer devoid of cells of hematopoietic origin. Addition of IL7 to such cocultures further increased BCP proliferation. BCP were shown to proliferate as stroma-adherent and non adherent cells. Total cell numbers expanded during 3 weeks, as much as 8 fold in the presence of IL7 when compared with input BCP numbers. Finally, BCP remained sIgM- in stroma dependent cultures, and only a subpopulation of cells became CD20+ in the presence of IL7. Our present study demonstrates the feasibility of human BCP expansion in vitro. However, the signals required for the transition of BCP to mature B cells remain to be determined.

In vitro growth and maturation of human B-cell precursors.

Purified B cell precursors (BCP) (CD10+ CD19+ surface-membrane (s)Ig-cells) isolated from human fetal bone marrow (BM) were cultured with various cytokines, in the presence or absence of a fibroblastic stromal cell layer derived from adult human BM. We demonstrated that IL-7, IL-3, and stem-cell factor (SCF) participate in inducing low magnitude BCP proliferation in the absence of stroma. Addition of either IL-4, IFN (alpha and gamma), or TGF beta, resulted in significant inhibition of proliferation. Strikingly, BCP proliferated at remarkably higher levels when cultured on BM stromal cells, and this effect was further enhanced by exogenously supplied IL-7. Proliferating cells were mostly CD20+, and included both c mu- and c mu+ cells. Furthermore, BCP proliferated in response to anti CD40 antibody presented by Fc gamma RII-transfected murine fibroblastic Ltk- cells (CD40 system) (Banchereau et al. 1991), demonstrating a functional role for CD40 in B cell ontogeny. However, this effect was shown to require a second signal, which could be specifically provided by IL-3 among a panel of cytokines examined. Finally, although suggestive of BCP maturation, the culture systems examined did not permit the transition to mature B cells (sIgM+ sIgD+).

Distribution of surface-membrane molecules on bone marrow and cord blood CD34+ hematopoietic cells.

We have investigated the distribution of membrane molecules on CD34+ hematopoietic cells isolated from human bone marrow (BM) and cord blood (CB). A distinct CD10+ population was present in BM, but it was not detected in CB. Most CD34+ CD10+ cells in BM were B-cell precursors (BCP), because they expressed CD19. However, CD40 and CD37 were found on the majority of CD34+ cells from either BM or CB, demonstrating that these antigens are not restricted to B-lineage CD34+ cells. CD40 and CD37 were lost during culture of CD34+ cells in the presence of interleukin 3 (IL-3), indicating transient expression early in myeloid development. CD13 antigen was detected on virtually all CD34+ cells from BM and CB. Accordingly, CD13 was present on CD34+ CD10+ cells, demonstrating that this structure is not restricted to myeloid CD34+ cells. In contrast, myeloid CD33 antigen was not detected on CD34+ CD10+ cells. Expression levels of CD13 and of CD33 were heterogeneous in BM, reflecting diversity within the resident CD34+ population. CD25 and CD71 were found on a proportion of CD34+ cells from either BM or CB and maintained during culture in IL-3, consistent with a distribution on activated cells. Finally, a variety of adhesion receptors were present on CD34+ cells. These included the alpha 4 beta 1 (VLA-4), alpha 5 beta 1 (VLA-5), and alpha L beta 2 (LFA-1) integrins, as well as ICAM-1, LFA-3, H-CAM, and LAM-1. Expression of adhesion receptors was remarkably similar in BM and CB, and it followed an all-or-nothing pattern that failed to delineate CD34+ subsets. Taken together, our data show that although CD34+ cells from BM constitute a more heterogeneous population, resident and circulating CD34+ cells largely display the same cell-surface molecules.

Interleukin-7 induces the proliferation of normal human B-cell precursors.

In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL-7 acts as a growth factor during the ontogeny of human B lymphocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures: endogenous production in the adherent layer and effect of exogenous GM-CSF on granulomonopoiesis.

This study was designed to assess the presence of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) within adherent layers of human Dexter-type cultures and to investigate the effect on granulomonopoiesis of adding exogenous GM-CSF to the culture medium. The presence of GM-CSF was demonstrated using a bioassay, in which adherent layers from normal bone marrows gave rise to endogenous granulocyte-macrophage colony-forming units (CFU-GM) that were specifically inhibited by increasing amounts of an anti-GM-CSF neutralizing antibody. Using an immunoassay, the estimated amounts of GM-CSF were less than or equal to 40 pg per flask in adherent layers, while remaining undetectable in supernatants. The addition of 10 ng or purified recombinant GM-CSF per milliliter of culture medium increased slightly the CFU-GM output over a 5-week culture period. The addition of 50 ng/mL decreased significantly the CFU-GM output after 5 weeks of culture. This decrease was associated with major modifications of the adherent layer cell composition. Large round or ovoid macrophages were generated at the expense of the interdigitated and elongated stromal cells and the extracellular fibronectin network was no longer observed. These studies suggest that GM-CSF production by accessory cells (stromal cells and/or monocytes) is almost equal to its consumption by hematopoietic cells, a situation similar to that found in long-term cultures of murine marrows. They also show that the maintenance of granulomonopoiesis is decreased by adding more than 10 ng/mL of exogenous GM-CSF to the culture medium, which is related to the induction of adherent macrophages, the disappearance of the major smooth-muscle-like stromal cell component of the adherent layer, and that of the fibronectin extracellular matrix.

Potentiation of early hematopoiesis by tumor necrosis factor-alpha is followed by inhibition of granulopoietic differentiation and proliferation.

We have previously shown that tumor necrosis factor-alpha (TNF alpha) strongly potentiates interleukin-3 (IL-3)-induced short-term proliferation of human CD34+ hematopoietic progenitor cells (HPC). Using longer term cultures of CD34+ HPC, we demonstrate here that this initial potentiation ceases after 10 to 12 days; whereupon TNF alpha displays inhibitory effects. Thus, TNF alpha was found to inhibit cells of granulocytic affiliation while it potentiates the development of maturing cells of the monocytic lineage both in liquid and semi-solid (day 14 colony-forming unit) cultures. TNF alpha was demonstrated to reversibly block granulocytic differentiation at the level of uncommitted CD13-, CD15- blast cells that accumulate in IL-3 + TNF alpha cultures. Furthermore, growth of committed granulocytes (CD15+) from IL-3 cultures was also inhibited by TNF alpha through an arrest of cell cycle in G0/G1. Finally, the use of neutralizing anti-TNF alpha monoclonal antibody and limiting dilution studies indicate that the inhibitory effects of TNF alpha are direct. Taken together, our data demonstrate that, following a phase of potentiation of proliferation of early HPC, TNF alpha displays direct inhibitory effects due to negative interference with both granulocytic differentiation and proliferation of granulocytic cells.