Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.     

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.     

Role of granulocyte/macrophage colony-stimulating factor in the regulation of murine alveolar macrophage proliferation and differentiation

Granulocyte/macrophage (GM)-CSF is one of the hemopoietic growth factors that stimulates neutrophilic granulocyte and macrophage production by bone marrow progenitor cells. In this study, the effect of GM-CSF on the growth and differentiation of murine pulmonary alveolar macrophages (PAM) was investigated. In the presence of GM-CSF, normal murine PAM were induced to proliferate and develop into macrophage colonies with a dose-response curve similar to that of bone marrow GM colony-forming cells. PAM also responded to CSF-1, a lineage-restricted growth factor, but required much higher doses of CSF-1 and a longer incubation time for optimal colony formation. The proliferative response of PAM to CSF-1, however, was greatly enhanced by the concurrent addition of low doses of GM-CSF. In contrast, low doses of CSF-1 failed to potentiate the proliferative response of PAM to GM-CSF. Macrophages derived from GM-CSF cultures were rounder and less stretched and possessed less FcR-mediated phagocytic activity than cells produced in CSF-1 cultures. A study with hydrocortisone-induced monocytopenia showed that nearly one half of lung macrophages may be sustained by local proliferation of PAM without the continuous migration of blood monocytes. This study suggests that GM-CSF may play a major role in the production of PAM by two modes of action, 1) direct stimulation of cell proliferation and 2) enhancement of their responsiveness to CSF-1, thereby producing more mature and functionally competent macrophages.     

CSF-1 stimulates nucleoside transport in S1 macrophages

We have examined nucleoside transport (NT) in a cell line derived from primary day 7 murine bone marrow macrophages (S1 macrophages) in response to the macrophage growth factor, colony-stimulating factor 1 (CSF-1). Adenosine and uridine transport in quiescent S1 macrophages occurred primarily by two facilitated diffusional routes, one that was sensitive and one that was relatively resistant to the inhibitor nitrobenzylthioinosine (NBMPR). Addition of CSF-1 to quiescent cultures resulted in increased adenosine and uridine transport with biphasic kinetics with respect to the cell cycle. Basal NT activity was elevated (about twofold) within 15 min of CSF-1 addition, returned to near basal levels by 1 h, and then increased again (three- to fourfold) 8–12 h later, returning again to basal levels by 48 h post CSF-1 stimulation. We propose that the large increase in NT activity at 8–12 h corresponded with the time when cultures synchronously began to enter the S phase of the cell cycle. In addition to these changes in the absolute rates, the proportions of NBMPR-sensitive and NBMPR-insensitive transport also change after CSF-1 addition. Quiescent cultures exhibited primarily NBMPR-insensitve transport while logrithmically growing cultures exhibited primarily NBMPR-sensitive nucleoside transport activity. The increase in the NBMPR-sensitive component of the transport process paralleled a similar increase in the number of high-affinity NBMPR binding sites, suggesting that the mechanism for upregulating NBMPR-sensitive NT activity involves increases in the number of NBMPR-sensitive transporter sites. Interestingly, we were unable to detect Na+-dependent concentrative uptake of adenosine, uridine, or formycin-B either in the S1 macrophage cell line or in primary (day 7) murine macrophages. Thus these bone marrow derived macrophages did not display the characteristically large Na+-dependent transport systems observed by others in peritoneal macrophages, implying that these two populations of macrophages are, indeed, functionally distinct. © 1993 Wiley-Liss, Inc.     

Type-beta transforming growth factor inhibits proliferation and expression of alkaline phosphatase in murine osteoblast-like cells

TGF-beta modulates growth and differentiation in many cell types. MC3T3E1 is a clonal non-transformed murine bone cell line which differentiates in culture. We tested the effect of porcine TGF-beta on the proliferation and differentiation of MC3T3E1 cells in monolayer cultures by following cell number, and alkaline phosphatase activity. TGF-beta treatment (2 ng/ml) altered the shape of MC3T3E1 cells from cuboidal to elongated/spindle-shape. TGF-beta inhibited the growth of MC3T3E1 by up to 40% (P less than 0.02) in a dose-dependent manner with half maximal inhibition at 1 ng/ml. Growth inhibition depended on serum concentration, maximal inhibition occurring at 2% serum. Expression of alkaline phosphatase, which peaks in vitro when the cells reach confluence, was strongly inhibited by TGF-beta, in a dose-dependent manner with half maximal inhibition at around 0.05 ng/ml and complete inhibition at 2 ng/ml. Alkaline phosphatase inhibition was irreversible after 24 hours exposure to TGF-beta.     

Bradykinin sensitization of CSF-1-responsive murine mononuclear phagocyte precursors to prostaglandin E

Bradykinin (BK) inhibited clonal proliferation of CSF-1-stimulated mononuclear phagocyte precursors derived from murine marrow. This inhibitory effect of BK was restricted to the subpopulation of precursors that required two signals, CSF-1 and LPS, for clonal proliferation. No effect was observed on stimulated colony formation by precursors that responded solely to the addition of CSF-1. Inhibition of colony formation by the two signal-dependent precursors required the presence of adherent marrow cells and was mediated by endogenously produced PG. Although evidence was obtained indicating that BK augmented PG production by adherent cells, an additional effect of BK appeared to be a significant sensitization of the two signal-dependent precursors to inhibition by PGE.     

Bone marrow-derived macrophages: development and regulation of differentiation markers by colony-stimulating factor and interferons

To investigate the role of specific cytokines in the development of the fully mature macrophage, we have employed murine bone marrow cells that were grown in the presence of CSF-1, a colony-stimulating factor that has been shown to induce the proliferation and differentiation of macrophages from their precursor cells. The CSF-1 employed in these studies was partially purified to ensure removal of contaminating interferon (IFN) from the preparations. After 1 to 2 wk in the presence of the partially purified CSF-1, the adherent macrophages were removed from flasks enzymatically and were recultured at known densities in the absence of CSF-1. Cell surface antigens (Mac-1 and Ia) and Fc receptor capacity (as assessed by Fc-mediated phagocytosis) were examined as markers of macrophage differentiation. Basal levels of Fc receptor capacity and Mac-1 antigen were markedly influenced by exposure to CSF-1, and appear to be modulated by CSF-induced, macrophage-derived IFN. When the bone marrow-derived macrophages were exposed to exogenous IFN in the absence of CSF-1, they proved to be extremely inducible with respect to Fc-mediated phagocytosis (IFN-beta and rIFN-gamma) and Ia antigen expression (rIFN-gamma) when compared with thioglycollate-elicited macrophages. Thus, macrophage growth factors, such as CSF-1, promote macrophage maturation by inducing the production of autostimulatory signals, such as macrophage-derived IFN. In addition, exogenous cytokine stimuli, such as IFN-gamma, further amplify the differentiative potential of these cells. Bone marrow-derived macrophages, propagated under well-defined conditions and never exposed to eliciting agents, provide a powerful model for studying the role of cytokines, such as CSF-1 and IFN, in the differentiative pathway of macrophages.     

Effects of transforming growth factor beta and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells

When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na beta-glycerophosphate, discrete three-dimensional nodules form with the histologic, immunohistochemical, and ultrastructural characteristics of bone (Bellows et al; Calcified Tissue International 38:143-154, 1986; Bhargava et al., Bone, 9:155-163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8-13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF-beta) results in dose-dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2-200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty-eight hour pulses of TGF-beta (0.01-1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF-beta and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF-B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation.     

The nature of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF-1 binding to macrophages

The tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF-1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil-neutrophil, neutrophil, neutrophil-macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum-free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA-stimulated colony formation was suboptimal and delayed in serum-free cultures that could support optimal colony formation in the presence of CSF-1. In addition, TPA did not directly compete with [125I]CSF-1 at 4 degrees C for its specific, high-affinity receptor on mouse peritoneal exudate macrophages. However, a 2-hour preincubation of the cells with TPA at 37 degrees caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF-1 in its effects on CSF-1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down-regulate the CSF-1 receptor) but it is not able to mimic CSF-1 in other ways (TPA alone cannot stimulate the full CSF-1 response, TPA does not stimulate the most primitive CSF-1 responsive cells, and TPA does not bind to the CSF-1 receptor).     

Bradykinin sensitization of colony-stimulating factor-1-responsive murine marrow progenitors to prostaglandin E. A property of the amino-terminal tetrapeptide fragment

The active sequence in bradykinin (BK) responsible for PGE-aided inhibition of CSF-1-stimulated clonal proliferation of murine mononuclear phagocyte progenitors was determined. In total marrow cultures, BK and (D-Phe7)-BK, a specific BK antagonist, inhibited colony formation by CSF-1 responsive precursors that require two signals, CSF and LPS, for clonal proliferation. (Lys1)-BK, an inactive BK analogue with Lys substituted for the amino-terminal Arg, was inactive. Arg-Pro-Pro-Gly, the amino-terminal tetrapeptide fragment of BK, was fully capable, on a molar basis, of replacing either BK or (D-Phe7)-BK as an inhibitor. Bk, (D-Phe7)-BK, and Arg-Pro-Pro-Gly were not inhibitory for colony formation in cultures containing indomethacin or in cultures depleted of adherent marrow cells. However, in these cultures addition of 10(-9) M PGE2 fully restored inhibition of two-signal-dependent colony formation. PGE2-dependent inhibition by the three peptides was equivalent on a molar basis indicating that Arg-Pro-Pro-Gly contains the sequence responsible for this inhibitory effect of BK and is sufficient to exert PGE-dependent inhibition of two-signal-dependent colony formation. The two-signal-dependent progenitors appear to be in transition to CSF competence suggesting that BK and PGE produced in an hematopoietic environment may act together to limit the production of new macrophages by inhibiting progenitors in transition to CSF competence.     

Development and characterization of monoclonal antibodies to murine macrophage colony-stimulating factor

The macrophage-specific CSF (CSF-1), purified from murine L cell-conditioned medium, supports the in vitro proliferation and survival of various murine mononuclear phagocyte colony-forming cells. In this report we describe the production and functional characterization of two monoclonal antibodies (mAb) to CSF-1 obtained from rat X rat hybridomas. These two mAb are functionally distinct and recognize different epitopes on CSF-1. The mAb 5A1 binds to and inhibits the biologic function of CSF-1, and the second mAb (D24) binds CSF-1 but does not neutralize its biologic activity. The mAb 5A1 inhibits colony formation of tissue mononuclear phagocyte colony-forming cells as well as the committed bone marrow stem cells for both granulocytes and monocytes. The extent of colony inhibition by mAb 5A1 is dependent on the tissue origin of colony-forming cells. CSF-1 complexed with mAb 5A1 does not bind to its cell surface receptor of peritoneal exudate macrophages, and mAb 5A1 does not complex with cell-bound CSF-1. Although both bone marrow cell-derived macrophages and J774.1 macrophages bind CSF-1, mAb 5A1 inhibits the proliferation of only bone marrow cell-derived macrophages. The non-neutralizing mAb D24 does not block binding of CSF-1 to its cellular receptor, and it recognizes cell-bound CSF-1.     

Inhibition of hemopoiesis in murine marrow cell cultures by recombinant murine tumor necrosis factor alpha: evidence for long-term effects on stromal cells

The addition of recombinant murine tumor necrosis factor alpha (rmTNF-alpha) to serum-free methylcellulose cultures inhibited macrophage colony formation stimulated by purified colony stimulating factor-1 (CSF-1), recombinant granulocyte-macrophage-CSF (rmGM-CSF), and recombinant interleukin 3 (rmIl-3). The concentration of rmTNF-alpha inhibiting colony formation by 50% (IC50) was between 2 and 20 ng/ml. Erythroid colony formation in cultures with erythropoietin (EPO) alone or EPO, rmIl-3, and rmGM-CSF in combination were reduced to a much lesser extent. In established long-term marrow cultures (LTMC), addition of 20 and 200 ng/ml of rmTNF-alpha resulted in release of cells from the adherent layer during the first week. Treatment of cultures with rmTNF-alpha for 4 consecutive weeks led to prolonged inhibition of cell production lasting up to 8 weeks after cessation of treatment. One day after addition of a low dose of TNF (2 ng/ml), "fat" cells were no longer observed in the adherent layer. Our results indicate that TNF inhibition of hemopoiesis occurs both at the progenitor cell and stromal cell levels.     

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.     

Persistent production of colony-stimulating factor (CSF-1) by cloned bone marrow stromal cell line D2XRII after X-irradiation

The adherent stromal layer in long-term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony-stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 X 10(6) D2XRII cells in 8.0 ml produced CSF-1 (M-CSF) at around 100-150 units/0.1 ml medium. Following X-irradiation there was a dose-dependent decrease in the production of CSF-1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X-ray dose reducing CSF-1 production to 50% was 100-fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X-irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF-1. The data indicate significant divergence of two biologic effects of X-irradiation on plateau-phase marrow stromal cells: physiologic function of adherence and CSF-1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X-irradiation-associated marrow suppression and leukemogenesis.     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Interactions among granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IFN-gamma lead to enhanced proliferation of murine macrophage progenitor cells

This report examines the actions of IFN-gamma on monocytopoiesis in murine liquid and semisolid bone marrow cultures. The proliferative response of bone marrow cells to macrophage CSF and granulocyte-macrophage CSF was assayed by measuring [3H]TdR uptake in a range of mouse strains. No interstrain difference in kinetics was observed for CSF-1 action, but GM-CSF acted significantly more rapidly on C57B1/6, Swiss, and to a lesser extent A/J mice than on BALB/c or CBA. IFN-gamma inhibited [3H]TdR incorporation elicited by CSF-1, and to a much lesser extent, GM-CSF. When the two CSF were added together, the effects were not additive; in fact, the response was the same as that seen with GM-CSF alone. When IFN-gamma was also added, the response was restored to the level seen with CSF-1 alone. In essence, the inhibitory actions of GM-CSF and IFN-gamma were mutually exclusive. The mechanism of these actions was investigated using colony assays. As expected, CSF-1 caused the formation of pure macrophage colonies, whereas GM-CSF stimulated production of macrophage, granulocyte, and mixed granulocyte macrophage colonies. When the two CSF were added in combination, the total colony count was greater than with either alone, but less than additive. The number of pure macrophage colonies was reduced to the number seen with GM-CSF alone. IFN-gamma reduced the number of colonies in the presence of CSF-1, but slightly increased the number with GM-CSF. In the presence of both CSF, IFN-gamma increased the colony count by around 25 to 40%, so that the numbers were greater than the combined total of CSF-1 plus GM-CSF added separately. Similar results were obtained in all mouse strains tested. The results suggest that the thymidine uptake data reflect changes in the number of progenitor cells responding rather than changes in cell cycle time. The results are discussed in terms of the possibility that coadministration of GM-CSF and CSF-1 could ameliorate the myelosuppressive actions of IFN-gamma in vivo, leading to more effective use of this agent as a biologic response modifier.     

Transforming growth factor-beta 1 selectively inhibits IL-3-dependent mast cell proliferation without affecting mast cell function or differentiation

Transforming growth factor-beta 1 (TGF-beta 1) is an important regulator of cell growth, differentiation, and function. We show that TGF-beta 1 selectively inhibits IL-3-dependent mouse bone marrow derived mast cell (MBMMC) proliferation without affecting MBMMC function or differentiation. TGF-beta 1 significantly decreased [3H]thymidine uptake by IL-3-dependent MBMMC in a dose-dependent manner with 50% inhibition of proliferation occurring with a TGF-beta 1 concentration of 0.1 ng/ml. A brief (i.e., 30 min) incubation of MBMMC with TGF-beta 1 is sufficient to inhibit IL-3-induced proliferation of MBMMC (cultured in the absence of TGF-beta 1) for 24 to 48 h. The inhibitory effect of TGF-beta 1 on the IL-3-dependent proliferation of MBMMC is not cytotoxic as evident from the absence of MBMMC trypan blue staining, the retained functional characteristics of the MBMMC cultured in TGF-beta 1, and the reversibility of the TGF-beta 1 induced inhibition of IL-3 dependent MBMMC proliferation. MBMMC grown in TGF-beta 1 acutely (24 to 48 h) or chronically (7 to 14 days) do not exhibit functional differences in performed or newly generated mediator secretion (Ag/IgE or calcium ionophore A23187 induced MBMMC beta-hexosaminidase or leukotriene C4 release) from MBMMC grown in the absence of TGF-beta 1. In addition, MBMMC cultured for 2 wk in TGF-beta 1 do not show evidence of differentiation as assessed by cellular histamine content or Alcian blue/safranin staining. Thus, TGF-beta 1 is an important negative regulator of IL-3-dependent mast cell proliferation in vitro, selectively inhibiting IL-3-dependent MBMMC proliferation without affecting MBMMC function or differentiation.