Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.     

Transforming growth factor-beta inhibits the production of IgG, IgM, and IgA in human lymphocyte cultures.

Transforming growth factor beta (TGF beta) has potent immunoregulatory effects acting on both T and B cells. It strongly inhibits secretion of IgG and IgM in human and murine B cell cultures, but has been shown to have an enhancing effect on IgA production in the mouse. We have studied the effect of TGF beta on the production of IgA in human lymphocyte cultures. The addition of TGF beta to pokeweed-stimulated peripheral blood lymphocytes resulted in a suppression of IgA production of both subclasses, similar in magnitude to the suppression of IgG and IgM production. Membrane IgA expression was not increased by culturing tonsillar lymphocytes with TGF beta. In conclusion, we find no evidence for a selective enhancing effect of TGF beta on IgA synthesis in humans, in contrast to the findings reported in mice.     

Modulation of interleukin-1 beta production by cyclic AMP in human monocytes

Elevation of cAMP has been considered to be an important downregulative signal in the production of interleukin-1(IL-1). This study demonstrates that this phenomenon is dependent on the signal used to activate the IL-1 production. The IL-1 beta production of lipopolysaccharide activated human monocytes was readily inhibited by dibutyryl cAMP. This took place without a significant change in the steady-state levels of IL-1 beta mRNA. By contrast, in PMA activated monocytes 100 microM dibutyryl cAMP increased in IL-1 beta production ca. 4-fold. The steady-state levels of IL-1 beta mRNA were also simultaneously increased.     

Transforming growth factor-beta enhances the M-CSF and GM-CSF-stimulated proliferation of macrophages.

Transforming growth factor-beta (TGF-beta) has been shown to regulate the proliferation and function of several different cell types in the immune system. We have examined the effect of TGF-beta on the proliferation of murine macrophages in liquid culture. TGF-beta by itself did not induce proliferation of differentiated (7 days in culture) bone marrow-derived macrophages (BMM). In the presence of M-CSF, TGF-beta enhanced the proliferation of differentiated BMM and elicited peritoneal macrophages but had an inhibitory effect on the proliferation of nonadherent BMM (3 days in culture). The effect of TGF-beta was not restricted to M-CSF-dependent proliferation but was also observed for GM-CSF-dependent proliferation. The autocrine production of TGF-beta appeared to contribute to the proliferation of BMM. The addition of antibody against TGF-beta inhibited M-CSF- and GM-CSF-dependent proliferation 32% and 28%, respectively. In bone marrow, TGF-beta may be an important negative regulator of macrophage proliferation; whereas, in the tissues, TGF-beta may enhance macrophage proliferation.     

Cells derived from human umbilical cord blood support the long-term growth of undifferentiated human embryonic stem cells

Various types of human cells have been tested as feeder cells for the undifferentiated growth of human embryonic stem cells (hESCs) in vitro. We report here the successful culture of two hESC lines (H1 and H9) on human umbilical cord blood (UCB)-derived fibroblast-like cells. These cells permit the long-term continuous growth of undifferentiated and pluripotent hESCs. The cultured hESCs had normal karyotypes, expressed OCT-4, SSEA-4, TRA-1-60, and TRA-1-81, formed cystic embryonic body in vitro and teratomas in vivo after injected into immunodeficient mice. The wide availability of clinical-grade human UCB makes it a promising source of support cells for the growth of hESC for use in cell therapies.     

Transforming growth factor-beta 1 enhances the suppression of human hematopoiesis by tumor necrosis factor-alpha or recombinant interferon-alpha

The effects of transforming growth factor-beta 1 (TGF-beta 1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor-alpha (TNF-alpha) and recombinant human interferon-alpha (rIFN-alpha). Combinations of TNF-alpha and TGF-beta 1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU-E) and an additive suppression of granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. In addition, TGF-beta 1 synergized with rIFN-alpha to suppress CFU-GM formation, while the combined suppressive effects of both cytokines on CFU-GEMM and BFU-E were additive. When TGF-beta 1 was tested with TNF-alpha or IFN-alpha on granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells in a 5-day proliferation assay, the antiproliferative effects of TGF-beta 1 and TNF-alpha were additive, while those with TGF-beta 1 and rIFN-alpha were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG-1 where TGF-beta 1 in combination with TNF-alpha resulted in an additive suppression while inhibition by TGF-beta 1 and IFN-alpha was synergistic. These results demonstrate for the first time the cooperative effects between TGF-beta and TNF-alpha and IFN-alpha in the suppression of hematopoietic cell growth, raising the possibility that TGF-beta might be used in concert with TNF-alpha or IFN-alpha in the treatment of various myeloproliferative disorders.     

Primary cultures of epithelial cells and long-term cultures of fibroblasts from the human umbilical cord: ultrastructural and immunocytochemical characterization

Repeated trypsinization of the full term human umbilical cord epithelium allows an homogeneous and exclusively epithelial primary culture, without fibroblastic growth. Transmission electron microscopy observations of desmosomes, cytokeratin intermediate filaments as revealed by indirect immunofluorescence and cultural aspects confirm the epithelial nature of this primary culture. Fibroblasts obtained by an explants culture method exhibit neither desmosomes nor cytokeratin intermediate filaments, which are epithelial markers. They yield characteristic long term cultures of fibroblastic aspect and growth.     

Transforming growth factor-beta regulates the expression of luteinizing hormone receptors in ovarian granulosa cells

In cultured granulosa cells, addition of 1 to 50 ng follicle-stimulating hormone induced a 350-fold rise in luteinizing hormone receptors, while larger amounts of gonadotropin up to 200 ng reduced these receptors to approximately 50% of peak levels. Transforming growth factor-beta (16 pM) enhanced the stimulatory actions of low levels of gonadotropin (2.5-10 ng) by 2 to 3-fold, and inhibited the induction of luteinizing hormone receptors by higher levels of follicle-stimulating hormone (greater than or equal to 50 ng) by 30-50%. The actions of the growth factor were concentration-dependent over the range from 0.8 to 16 pM and included a similar biphasic effect upon gonadotropin-induced cAMP production. Modulation of cAMP formation and luteinizing hormone receptor expression by transforming growth factor-beta could influence the ability of the granulosa cell to respond to luteinizing hormone during ovarian follicular maturation and ovulation.     

Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production.

Isolated rat hepatocytes in primary monolayer culture were maintained for 18-24 h in the presence of 10% (v/v) serum and [3H]inositol. Vasopressin (100 nM) stimulated the production of inositol mono-, bis- and tris-phosphates (IP1, IP2, and IP3). Prior exposure of hepatocytes to 8-bromo cyclic AMP (8Br-cAMP; 100 microM), but not 8-bromo cyclic GMP, enhanced the vasopressin-mediated stimulation of inositol phosphate accumulation, but had no significant effect on their formation in the absence of vasopressin. The effect of the cyclic AMP analogue was mimicked by glucagon (10 nM), and was seen whether cyclic AMP or glucagon was added 5 min or 12 h before the addition of vasopressin. An 8 h incubation with dexamethasone (100 nM) enhanced the accumulation of IP3, but not that of IP2 or IP1, in the presence of 8Br-cAMP and vasopressin. Cycloheximide or actinomycin D had little effect on the vasopressin stimulation of inositol phosphate accumulation, after an 8 h incubation in the presence or absence of 8Br-cAMP.     

Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts.

Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.     

A serum-free medium formulation supporting growth of human umbilical cord vein endothelial cells in long-term cultivation

A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences in final cell density compared to controls cultivated with serum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transforming growth factor-beta enhances the in vivo effector function and memory phenotype of antigen-specific T helper cells in experimental autoimmune encephalomyelitis.

Transforming growth factor-beta (TGF-beta) had a profound effect on the in vitro phenotypic development of Ag-activated Th cells and enhanced the in vivo effector function of these cells upon adoptive transfer. Previous studies have shown that there are two types of Th cell populations found in unimmunized animals, naive helper cells, which are short-lived and express low levels of CD44 and high levels of CD45R and Mel-14, and memory helper cells, which have a long life span and express high levels of CD44 and low levels of CD45R and Mel-14. Culturing of Ag-specific murine Th cell lines and clones in the presence of TGF-beta greatly enhanced both the memory phenotype of the cultured cells and the effector function upon adoptive transfer in experimental autoimmune encephalomyelitis. Histologic evaluation of spinal cords from recipients receiving passively transferred murine T cell lines cultured with TGF-beta revealed large demyelinated plaques (multiple sclerosis-like) that were not present in animals receiving cells cultured with Ag alone. TGF-beta also enhanced the capability of myelin basic protein-specific Lewis rat T cell lines to transfer experimental autoimmune encephalomyelitis and potentiated a purified protein derivative-specific rat helper cell line to transfer delayed type hypersensitivity. Thus, the effects of TGF-beta did not appear to be limited by species specificity, Ag specificity, or in vivo T cell function. This is the first study showing that TGF-beta can potentiate the development and maintenance of the Th cell memory phenotype in vitro and enhance their in vivo effector function in an animal disease model.     

Murine survival of lethal irradiation with the use of human umbilical cord blood.

We have found that human umbilical cord blood (HUCB) will routinely protect mice exposed to lethal levels of irradiation. At the end of 50 days, over seventy percent (70%) of mice injected with HUCB survived 900 cGy or irradiation, which produced 100% deaths in the uninjected control animals. Moreover, there was some evidence that human colony stimulating factors further improved survival. Anti-Natural Killer cell (NK) antibody was utilized along with HUCB in these studies, however, Anti-NK cell serum alone had no radioprotective effect in mice. The studies reported here suggest the possibility of utilizing HUCB for immediate protection of humans from lethal irradiation.     

Ceramides, sphinganine, sphingosine and acid sphingomyelinases in the human umbilical cord blood.

Ceramides, sphingosine, sphinganine, as well as Zn (++)-dependent and Zn (++)-independent acid sphingomyelinase are present in the plasma of adults. The aim of the present study was to examine the concentrations of these compounds and activities of both enzymes in the umbilical cord blood in humans. Twenty-two women with uncomplicated term pregnancy volunteered for the study. Blood was taken from the umbilical cord artery and from the antecubital vein of the mother immediately after delivery. Free ceramides were isolated by thin layer chromatography, and their fatty acids were identified and quantified by gas-liquid chromatography. Free sphingosine and sphinganine concentrations were determined using high-performance liquid chromatography. Acid Zn (++)-dependent and Zn (++)-independent sphingomyelinase activity was measured using sphingomyelin [choline-methyl-14C] as a substrate. We found that the compounds examined are present in the umbilical cord blood. The total fatty acid-containing ceramide concentrations in fetal blood were lower than in mother's blood. The mean sphingosine and sphinganine concentrations in the fetal and maternal serum were similar. The examined enzymes were present in the fetal serum, and their mean activity did not differ from that in the mother. In conclusion, we have shown the presence of ceramides, sphingosine and sphinganine and both isoforms of acid sphingomyelinase in the human fetal cord blood. They are most likely the product of the fetus itself.     

Transforming growth factor beta induces the production of interleukin 6 by human peripheral blood mononuclear cells

Previous studies have indicated that the cytokine transforming growth factor beta 1 (TGF beta 1) has immunosuppressive properties and can inhibit the production of tumor necrosis factor (TNF) and Interleukin 1 (IL 1) by human peripheral blood mononuclear cells. In this study, we have examined the effects of TGF beta 1 on the production of Interleukin 6 (IL 6) by human peripheral blood mononuclear cells. Treatment with only TGF beta 1 leads to the induction of IL 6, and this was both dose- and time-dependent. The effect of TGF beta 1 was evident at the level of IL 6 mRNA, suggesting TGF beta 1-induced de novo synthesis of IL 6. Induction of IL 6 by TGF beta 1 was specific, as other cytokines made by mononuclear cells (TNF and IL 1) were not induced by TGF beta 1. Furthermore, when a panel of stimuli were compared for their ability to induce IL 1, TNF and IL 6 in the presence or absence of TGF beta 1, IL 6 levels were augmented in the presence of TGF beta 1, while the induction of IL 1 and TNF was inhibited significantly. These results indicate that TGF beta 1 has complex effects on the production of cytokines by peripheral blood mononuclear cells and that TGF beta 1 is not inhibitory for all cytokine production. The ability of TGF beta 1 to induce IL 6 suggests that IL 6 may mediate some of the effects of TGF beta 1.     

Transforming growth factor-beta inhibits phosphorylation of the retinoblastoma susceptibility gene product in human monocytic leukemia cell line JOSK-I.

Proliferation of the human monocytic leukemia cell line JOSK-I is inhibited by transforming growth factor-beta (TGF-beta). Growth inhibition by TGF-beta was not due to either a toxic effect or to induction of differentiation. TGF-beta induced a cell cycle arrest at late G1 phase and was not found to be inhibitory to JOSK-I cells in S phase or G2/M. This G1 cell cycle arrest was associated with an accumulation of the unphosphorylated form of the retinoblastoma susceptibility gene product (Rb) in good correlation with inhibition of DNA synthesis. In contrast to the effects of TGF-beta, two other agents which induced a G1 arrest of JOSK-I cells had a different effect on Rb. Aphidicolin blocked cells at G1/S but could not reduce Rb phosphorylation as great as that seen with TGF-beta. 12-O-Tetradecanoylphorbol-13-acetate, an inducer of differentiation, did reduce Rb phosphorylation, but not until 72 h, when differentiation had already occurred. The identities of the Rb kinases are unknown, but recent evidence suggests that the cdc2 gene product could participate in Rb phosphorylation. Although cdc2 mRNA and total protein levels were not affected, TGF-beta inhibited the rate of translation and kinase activity of cdc2 in JOSK-I cells. These results suggest that growth inhibition of hematopoietic cells by TGF-beta is linked to suppression of Rb phosphorylation to retain Rb in an unphosphorylated, growth-inhibitory state. The suppression of Rb phosphorylation is suggested to be mediated through inhibition of cdc2 kinase activity by TGF-beta.