Effect of cytokines on proliferation of Epstein Barr virus-transformed B lymphocytes

Plating efficiencies of EBV transformed human B cells seeded in single cell cultures are far lower (less than 1%) than observed in T cell cloning experiments. This report describes the stimulatory effect of several crude as well as recombinant growth factors on proliferation of EBV transformed B cells measured by [3H]thymidine uptake. Supernatant of LPS activated monocytes (HSF) and recombinant interleukin 4 (rIL-4), but not recombinant IL-1 beta, IL-2, IL-6, TNF alpha, GM-CSF, and interferon gamma increased [3H]thymidine incorporation. The combination of HSF and rIL-4 was found to be synergistic on B cell proliferation. Plating efficiency of EBV transformed B cells at limiting dilution was improved by HSF, but not by the combination of HSF and rIL-4.     

Influence of food deprivation and adrenal steroids on DNA synthesis in various mammalian tissues.

The DNA content of skeletal muscle increases as young rats grow. Food deprivation prevented this increase: total DNA remained constant, while muscle weight and RNA decreased. Diaphragms isolated from fasted rats incorporated [3H]thymidine into DNA far more slowly than tissues from fed rats. Incorporation returned to control levels on refeeding. Fasting for 24 or 48 h also markedly reduced [3H]thymidine incorporation by slices of liver, kidney, and brain. The factors responsible for this inhibition of DNA synthesis were investigated. Amino acids, insulin, or serum from fed or fasted rats failed to alter thymidine incorporation by muscle. Injection of hydrocortisone into normal rats reduced incorporation into kidneys, liver, and muscle within 4h. Incubation of hemidiaphragms with hydrocortisone suppressed [3H]thymidine incorporation within 2-3h. Adrenalectomy enhanced incorporation into DNA by diaphragm, liver, kidney, and brain. When fasted, adrenalectomized rats showed little or no suppression of [3H]thymidine incorporation and lost less weight than fasted controls. These data suggest that adrenal steroids are important in inhibiting DNA synthesis during normal growth and during fasting.     

Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitor of Interleukin 1 in Normal Human Urine

We investigated whether urine from normal individuals contains inhibitors of interleukin 1 (IL-1). Diafiltered urine from normal afebrile donors suppressed IL-1-induced interleukin 2 (IL-2) activity of mouse thymocyte supernatants. These supernatants, however, strongly suppressed IL-2-induced [3H]thymidine incorporation into the IL-2-sensitive cell line CTLL-2, whereas the urine preparations did not. This phenomenon was caused by an increased amount of thymidine secreted by the urine-treated thymocytes. Therefore, in order to prevent interference, experiments were carried out with excess [3H]thymidine. Under these circumstances, suppression of IL-1- and to a lesser extent IL-2-induced DNA synthesis was still observed, whereas the synergistic effect of IL-1 on IL-2-induced DNA synthesis was only marginally reduced. We conclude that suppression of IL-1-induced IL-2 production by mouse thymocytes is a major effect of the IL-1-inhibitory factor(s) in normal urine. When the murine EL4 cell line was used, the diafiltered urine failed to inhibit IL-1-induced IL-2 production. The detection of an IL-1 inhibitor in urine is therefore dependent on the target cells as well as the effects of IL-1 on these cells.     

The Effect of Mechanical Loading on the Metabolism of Growth Plate Chondrocytes

It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrix-forming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [³H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-³H]proline and [³⁵S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrix-forming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation.     

Effects induced by BMPS in cultures of human articular chondrocytes: comparative studies

We compared anabolic and anti-catabolic activities of selected bone morphogenetic proteins (BMP-2, -4, -6, and -7) and cartilage-derived morphogenetic proteins (CDMP-1 and -2) in human normal adult articular chondrocytes. Ankle chondrocytes were cultured in alginate beads in the presence of 10% serum and treated with either growth factors only (each at 100 ng/ml) or the combination of interleukin-1 (IL-1 beta) (0.1 ng/ml) and BMPs. Chondrocyte metabolism was assessed by proteoglycan (PG) synthesis, content, DNA content, and cell survival. The results showed that BMP-2, -4, and -7 were more potent in stimulating PGs than other growth factors tested. The highest levels of PG synthesis were detected at day 9 in the presence of BMP-7. With regard to anti-catabolic properties, the effect depended upon treatment scheme (simultaneous or sequential). Under simultaneous cultures, BMP-2, -4, and -6 failed to counteract IL-1 beta induced inhibition of PG synthesis, while the CDMPs restored this parameter to serum control levels. Only BMP-7 showed consistent and pronounced anti-catabolic activity in either culture treatment scheme. None of the factors induced cell death or chondrocyte proliferation. In conclusion, the growth factors tested showed different levels of effects on human chondrocytes in culture, but only BMP-7 displayed both strong anabolic and anti-catabolic properties.     

Establishment of a human B cell line that proliferates in response to B cell growth factor

A human B cell line which shows a marked dose dependence on B cell growth factor (BCGF) when cultured in less than or equal to 2% serum has been established. Human B lymphocytes were obtained from peripheral blood of normal donors and cultured in the presence of anti-IgM (mu chain specific) and BCGF. Frequent refeedings with fresh medium containing BCGF and anti-IgM led to the establishment of a long term cultured human B cell line, HAB-40. Phenotyping of HAB-40 revealed that the cell population consisted predominantly of IgM-bearing (72%) and B1 (100%) positive cells. This B cell line consistently secreted IgM and IgG when co-cultured in the presence of PMA, anti-IgM and beta or gamma interferon (IFN). Also, it was Epstein-Barr virus nuclear antigen (EBNA) positive (100%). HAB-40 cells have been successfully maintained in the presence of BCGF without anti-IgM for over a year. Removal of BCGF led to the rapid loss of viable cells in cultures containing less than 2% serum. HAB-40 cells in microassays exhibited a marked dose-dependent incorporation of [3H]thymidine in response to BCGF in the absence of any exogenous stimulants such as anti-IgM or Staphylococcus aureus Cowan I (SAC). Recombinant interleukin 2 (IL-2) failed to augment the [3H]thymidine uptake by these B cells despite the low density expression of Tac antigen (IL-2 receptor) on their cell surface, or even when the cells were stimulated with phorbol myristate acetate (PMA) to express higher density of Tac antigen (48%). HAB-40 cells could be maintained in BCGF which was partially purified to deplete it of other contaminating proteins. None of the seven well established EBNA-positive human B cell lines nor two chronic B lymphocytic leukemia (B-CLL) cell lines that were tested showed BCGF dependence. The same BCGF-active chromatographic fractions that were active on HAB-40 cells also stimulated BCL1 and normal human B cells stimulated with anti-IgM. In the presence of less than or equal to 2% serum proteins this cell line provides a simple, reproducible assay for BCGF even in the presence of contaminant IL-2.     

Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.     

Effects induced by BMPS in cultures of human articular chondrocytes: Comparative studies

We compared anabolic and anti-catabolic activities of selected bone morphogenetic proteins (BMP-2, -4, -6, and -7) and cartilage-derived morphogenetic proteins (CDMP-1 and -2) in human normal adult articular chondrocytes. Ankle chondrocytes were cultured in alginate beads in the presence of 10% serum and treated with either growth factors only (each at 100 ng/ml) or the combination of interleukin-1 (IL-1β) (0.1 ng/ml) and BMPs. Chondrocyte metabolism was assessed by proteoglycan (PG) synthesis, content, DNA content, and cell survival. The results showed that BMP-2, -4, and -7 were more potent in stimulating PGs than other growth factors tested. The highest levels of PG synthesis were detected at day 9 in the presence of BMP-7. With regard to anti-catabolic properties, the effect depended upon treatment scheme (simultaneous or sequential). Under simultaneous cultures, BMP-2, -4, and -6 failed to counteract IL-1β induced inhibition of PG synthesis, while the CDMPs restored this parameter to serum control levels. Only BMP-7 showed consistent and pronounced anti-catabolic activity in either culture treatment scheme. None of the factors induced cell death or chondrocyte proliferation. In conclusion, the growth factors tested showed different levels of effects on human chondrocytes in culture, but only BMP-7 displayed both strong anabolic and anti-catabolic properties.     

Hyperglycaemia inhibits thymidine incorporation and cell growth via protein kinase C,mitogen-activated protein kinases and nitric oxide in human umbilical vein endothelium

An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells (HUVECs). Cells in their log-phase of growth (3.7 +/- 0.3 days, n = 27) incubated for 30 min with 25 mM D-glucose, but not with equimolar concentrations of L-glucose or D-mannitol, exhibited reduced [3H]thymidine incorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cell volume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen-activated protein kinases (p42/44(mapk)), but inhibited superoxide dismutase (SOD). Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitor L-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. In the presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator phorbol 12-myristate 13-acetate (PMA), the NO donor S-nitroso-N-acetyl-L,D-penicillamine (SNAP), dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A-23187. The effect of A-23187 was blocked by calphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.     

Reduced mitogen stimulation of DNA synthesis in human lymphocytes by a human recombinant interleukin-1 receptor antagonist.

The monokine interleukin-1 is produced by monocytes/macrophages after antigen/LPS stimulation and is an important early signal for the activation of resting T cells to become antigen specific T cells. However, little is known about the regulation and inhibition of IL-1. Recently, a new monokine has been described, generated by human macrophages, called interleukin-1 receptor antagonist (IL-1ra). This new monokine adheres to IL-1 in solution and blocks IL-1 receptor binding. IL-1ra is a glycoprotein structurally similar to IL-1 beta but having no interleukin-1-like activity. Using as a model mitogen (PHA 20 micrograms/ml)-stimulated lymphocyte DNA synthesis, we found that hrIL-1ra (30 min lymphocyte pretreatment) inhibits [3H]thymidine incorporation in a dose-dependent manner. This effect is most probably due to the inhibition of endogenous IL-1, which is a very important signal for T cell activation. The inhibition was maximum at the highest hrIL-1ra concentration used (250 ng/ml). However, when hrIL-1ra was added 2 h after PHA (20 micrograms/ml), a little, if any, inhibition of lymphocyte proliferation was found. The addition of hrIL-1ra simultaneously to the cell cultures with [3H]thymidine [( 3H]TdR) 6 h before the end of culture incubation did not significantly modify the results compared to the cells treated with PHA alone, indicating no interference of hrIL-1ra on [3H]TdR lymphocyte incorporation. We also found that the antibody anti-IL-1 beta inhibits mitogen stimulated lymphocyte DNA synthesis in dose-dependent concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the ratio of follicle-stimulating hormone to luteinizing hormone on rat granulosa cell proliferation and oestradiol-17 beta secretion.

The present study examined the effects of the ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) on granulosa cell proliferation and oestradiol-17 beta secretion. For these studies, ovarian segments from either immature rats or those primed with pregnant mares serum gonadotrophin (PMSG) were incubated for 5 h with [3H]thymidine and FSH (0-100 mIU/ml) with or without equivalent doses of LH. After incubation, granulosa cells were isolated and their mitotic activity estimated by determining the amount of [3H]thymidine incorporated into the DNA. The amount of oestradiol secreted into the media was measured by radioimmunoassay. Compared to granulosa cells from immature ovaries, granulosa cells from PMSG-primed ovaries required significantly less FSH to stimulate incorporation of [3H]thymidine, had a 9-fold higher basal level of oestradiol production and increased oestradiol secretion in response to gonadotrophins. At pharmacological serum levels (10-20 mIU of total gonadotrophin), FSH:LH ratios of less than or equal to 2 increased oestradiol secretion from PMSG-primed ovaries but did not increase the rate of [3H]thymidine incorporation. Conversely, FSH:LH ratios of greater than or equal to 3 stimulated [3H]thymidine incorporation without altering oestradiol secretion. These data demonstrate that granulosa cells of immature follicles not secreting oestradiol are relatively unresponsive to gonadotrophins at any dose tested. Once the capacity for oestradiol secretion develops, then both the dose and ratio of FSH and LH play major roles in determining whether the follicle will grow or secrete oestradiol.     

DNA synthesis in rabbit spleen explants. Its relationship to cell division.

The synthesis of DNA, as measured by the incorporation of [3H]-thymidine into the nuclei of cells of cultured explants of rabbit spleen, has been studied and correlated with estimation of DNA content of these nuclei using Feulgen densitometry, with and without colcemid arrest. Observations of the proportion of nuclei labelled with [3H]thymidine and their DNA content indicated that many cells were incorporating this precursor for DNA synthesis without the concomitant increases in DNA expected of cells in division cycle. Colcemid arrest failed to produce the changes in DNA content expected of a cell population as rapidly dividing as the labelling kinetics suggested. The results indicated that [3H]thymidine-labelled material, possibly DNA, was being transferred between cells and this accounted for the increased labelling index seen after pulsing with [3H]thymidine. The suppression of [3H]thymidine uptake by hydroxyurea indicated that this was not repair synthesis of damaged DNA.     

Activation and proliferation of normal resting human T lymphocytes in serum-free culture: role of IL-4 and IL-6

Purified human T lymphocytes, completely depleted of accessory cells [i.e. monocytes, large granular lymphocytes (LGL) and B lymphocytes], have been grown in serum-free culture in presence of a mitogenic lectin (phytohaemagglutinin, PHA) and different recombinant cytokines. Only IL-2 and IL-4 induced a marked stimulation of [3H] thymidine ([3H]TdR) uptake, cell proliferation and expression of activation markers [transferrin receptor (TrfR), IL-2R]. The other cytokines (IL-1 alpha, IL-1 beta, IFN-gamma, GM-CSF, TNF-alpha) had no significant effect, except for a moderate, but significant, stimulation of [3H]TdR uptake induced by IL-3. Simultaneous addition of IL-4 and anti-IL-2 neutralizing monoclonal antibodies (mAb) did not modify the effects induced by IL-4 alone. Furthermore, IL-2 was not detected in the supernatant of T cells grown in the presence of PHA and IL-4. Thus, our results indicate that IL-4 acts on T lymphocytes independently of IL-2. We also observed that IL-6 moderately activates DNA synthesis in PHA-stimulated T lymphocytes, but markedly potentiates the proliferative effect of suboptimal amounts of IL-2. In conclusion, the present study suggests that B-cell growth factors, in addition to IL-2, control the proliferation of normal circulating T lymphocytes.     

Decreased cell replication and polyamine content in insulin-producing cells after exposure to human interleukin 1 beta

Interleukin 1 (IL-1) has been suggested to cause the islet B cell destruction occurring during the development of insulin-dependent diabetes mellitus. One mechanism by which B cell loss can be compensated for is via de novo formation of new cells through replication. In the present study the replicatory activity of cells in isolated rat pancreatic islets and in the insulin-producing cell line RINm5F has been assessed by [3H]thymidine incorporation methods after exposure to 1-25 U/ml of human recombinant IL-1 beta (rIL-1 beta). In the rat islets [3H]thymidine incorporation was decreased by 20% 5 h after exposure to 25 U/ml rIL-1 beta. A similar inhibition was also observed in islets exposed to 2.5 and 12.5 U/ml rIL-1 beta. In the RINm5F cells there was a dose-dependent inhibition of the cell replication to approximately 50% of the controls in cells exposed to 25 U/ml rIL-1 beta for 48 h. This was also accompanied by an increased cell death, as measured by trypan blue inclusion (controls 13% and rIL-1 beta treated cells 25%). The insulin content of the RINm5F cells was reduced by about 40% after a 48-h exposure to 25 U/ml rIL-1 beta. When the exposure of the RINm5F cells to rIL-1 beta was decreased to 24 h there was no increased cell death, but a reduced replicatory activity was still observed. rIL-1 beta decreased the cellular content of the polyamines spermidine and spermine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple effects of human recombinant interleukin 4 on human myeloid monocyte cell lines

The effects of recombinant human interleukin 4 (rIL-4) on proliferation and differentiation on human myeloid/monocytic leukemia cell lines were examined. At high concentrations, rIL-4 had a slight enhancing effect on [3H]thymidine incorporation by U937 cells. rIL-4 markedly induced expression of the Fc epsilon receptor (CD23) and the Leu-M3 antigen (CD14) on U937 cells. HL60 and THP-1 cells treated with rIL-4 also showed increased CD23 expression, but little change of CD14 antigen expression. CD23 induction required lower amounts of IL-4 than needed for T cell growth, indicating that CD23 induction on U937 will serve as a sensitive assay for human IL-4. rIL-4 reduced the steady state level of IL-1 beta mRNA in U937.     

Effects of transforming growth factor-beta, transforming growth factor-alpha, and other growth factors on renal proximal tubule cells

Transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) addition to quiescent, confluent monolayers of rabbit renal proximal tubule cells in primary culture stimulated [3H]thymidine incorporation. TGF-alpha and EGF promoted a 14-fold rise in thymidine incorporation over control levels with half-maximal responses at 2 x 10(-9) M. IGF-1 only promoted a 4-fold rise in thymidine incorporation compared with control values with a half-maximal response of 10(-8) M. Platelet-derived growth factor alone did not stimulate [3H]thymidine incorporation and did not potentiate the effects of EGF or IGF-1 on DNA synthesis, suggesting that platelet-derived growth factor is neither a competence nor a progression growth factor for renal proximal tubule cells. TGF-beta inhibited both baseline and EGF-stimulated [3H]thymidine incorporation after 48 hours of exposure but enhanced EGF-stimulated DNA synthesis at 24 hours. Morphologic evaluation with phase contrast microscopy, scanning, and transmission electron microscopy demonstrated that TGF-beta promoted a dramatic phenotypic transformation of the epithelial monolayer with migration and adhesion of the cells to form solid clusters of adherent cells. Quantitative morphometry demonstrated that this transformation developed 24 hours after TGF-beta exposure, was nearing completion after 48 hours of TGF-beta treatment, and correlated to TGF-beta related inhibition of EGF-induced DNA synthesis (r = -0.82, p less than 0.01). These results demonstrate that EGF and TGF-alpha are the most potent growth promoters for renal proximal tubule cells. IGF-1 is only a modest growth promoter, whereas platelet-derived growth factor has no effect either as a competence or progressive growth factor. TGF-beta inhibited EGF-induced DNA synthesis but only after observable phenotypic transformation of the cells. The degree of TGF-beta promoted transformation on renal tubule cells was highly correlative to th e antiproliferative effect of TGF-beta, suggesting that similar molecular components which promote this phenotypic transformation may also be critical in the antiproliferative effect of TGF-beta.     

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.