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Results for “TGF-Beta pathway”

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PubMed1992

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.

Animals↗

PubMed1992

Leukemia inhibitory factor is expressed in cartilage and synovium and can contribute to the pathogenesis of arthritis.

This study reports on leukemia inhibitory factor (LIF) in human articular connective tissues. Biologically active LIF is present in synovial fluids from patients with osteoarthritis and at higher titers in samples from patients with rheumatoid arthritis. Cultured human synoviocytes and articular chondrocytes produced biologically active LIF and synthesized and secreted LIF proteins that migrated in SDS PAGE at approximately 43 kD. This was increased after stimulation with IL-1 beta. Chondrocytes in serum-containing cultures expressed the 4.2-kb LIF mRNA. IL-1 beta, LPS, and to a lesser extent tumor necrosis factor-alpha induced LIF gene expression. LIF autoinduced its mRNA and this provides evidence for an effect of this cytokine on function of joint tissue cells. Among a series of growth factors tested, transforming growth factor (TGF beta), including the isoforms TGF-beta1, TGF-beta 2, and TGF-beta 3, platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor induced this cytokine gene but differed with respect to the duration of their effects. Cultured synoviocytes expressed the LIF gene in response to the same set of peptide regulatory factors. Analysis of signal transduction pathways showed that PMA increased LIF mRNA, whereas calcium ionophore and cAMP had no detectable effects. Cycloheximide was a potent LIF mRNA inducer and dexamethasone inhibited LIF induced by PMA or IL-1 beta. Cartilage organ cultures and synovial tissues stimulated with IL-1 expressed high levels of LIF mRNA as demonstrated by in situ hybridization. These results identify LIF as a new cytokine that is produced by joint tissue cells and is overexpressed in arthritis. The induction of this cytokine by factors that are present during joint inflammation and the effects of LIF on connective tissue cells suggest that LIF is a mediator that can contribute to the pathogenesis of arthritis.

Arthritis↗

PubMed1992

Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts.

Transforming growth factor beta (TGF-beta), a multifunctional cytokine, is an indirect mitogen for human fibroblasts through platelet-derived growth factor (PDGF), particularly the A ligand-alpha receptor arm of that system. TGF-beta effects on PDGF alpha receptor expression were studied in vitro using ligand binding techniques in three human dermal fibroblast strains: newborn foreskin, adult skin, and scleroderma (systemic sclerosis, SSc). Each cell strain responded differently to TGF-beta. In newborn foreskin fibroblasts, PDGF alpha receptor number decreased in a dose-dependent manner after exposure to low concentrations of TGF-beta (0.1-1 ng/ml). Responses of normal skin fibroblasts were varied, and mean net receptor number was unchanged. Increases in PDGF alpha receptor number by TGF-beta occurred consistently with SSc fibroblasts and low concentrations of TGF-beta (0.1-1 ng/ml) were particularly stimulatory. Increased surface expression of alpha receptor subunit by TGF-beta in SSc fibroblasts correlated with increased new PDGF alpha receptor synthesis as demonstrated by radioimmunoprecipitation analysis of metabolically labeled cells and with increased steady-state levels of corresponding mRNAs. In normal adult skin fibroblasts, TGF-beta had no effect on either synthesis or mRNA expression of alpha receptor subunits. Proliferative responses to PDGF-AA after pretreatment with TGF-beta correlated positively with effects of TGF-beta on expression of alpha receptor subunit. Decreased mitogenic responses to PDGF-AA were observed in foreskin fibroblasts, small changes in responses in adult fibroblasts, and significant increases in SSc fibroblasts. Thus, costimulation with PDGF-AA and TGF-beta selectively enhanced proliferation of fibroblasts with the SSc phenotype. Immunohistochemical examination of SSc and control skin biopsies revealed the presence of PDGF-AA in SSc skin. Data obtained by ligand binding, immunoprecipitation, mRNA, and mitogenic techniques are consistent with the hypothesis that activation of the PDGF-AA ligand/alpha receptor pathway is a characteristic of the SSc fibroblast and may contribute to the expansion of fibroblasts in SSc.

Adult↗

PubMed1992

Regulation of leukaemic cells by interleukin 6 and leukaemia inhibitory factor.

Interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF) can have pleiotropic effects on different cell types. M1 myeloid leukaemic cells respond to IL-6 with activation of a terminal differentiation programme which includes activation of genes for certain haemopoietic regulatory proteins (IL-6, IL-1 alpha, IL-1 beta, granulocyte-macrophage colony-stimulating factor [GM-CSF], M-CSF, tumour necrosis factor and transforming growth factor [TGF] beta 1) and for receptors for some of these proteins, thus establishing a network of positive and negative regulatory cytokines. IL-6 and some other cytokines also induce during differentiation sustained levels of transcription factors that can regulate and maintain gene expression in the differentiation programme. M1 leukaemic cells induced to differentiate with IL-6 undergo programmed cell death (apoptosis) on withdrawal of IL-6, and can be rescued from apoptosis by IL-6, IL-3, M-CSF, G-CSF or IL-1, but not by GM-CSF. These differentiating leukaemic cells can also be rescued from apoptosis by the tumour promoter TPA (12-O-tetradecanoylphorbol-13-acetate) but not by the non-tumour-promoting isomer 4-alpha-TPA, and rescue from apoptosis can be achieved by different pathways. Apoptosis can also be induced in undifferentiated M1 leukaemic cells by expression of the wild-type form of the tumour suppressor p53 protein and IL-6 can rescue the cells from this wild-type p53-mediated apoptosis. There are clones of M1 cells that differentiate with IL-6 but not with LIF and another M1 clone that differentiates with either IL-6 or LIF. Differentiation induced by IL-6 or LIF is inhibited by TGF-beta 1. The pleiotropic effects of LIF, like those of IL-6, are presumably also in a network of interacting regulatory proteins.

Animals↗

PubMed1991

Cytokines and cell growth control.

Cellular proliferation in eukaryotic cells is associated with a highly organized sequence of events involving expression of growth-related genes. The growth regulatory cytokines such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF-I) are thought to be responsible for initiating proliferation of fibroblasts and other connective tissue cells. The immunomodulatory cytokines, including interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), are also known to modulate the growth response in nonimmune cells and regulate cellular metabolism by inducing expression of genes encoding biologically active molecules. While TGF-beta can induce anchorage-independent growth of nonneoplastic cells, it inhibits the proliferation of most normal cell types. Several of the cytokines that stimulate fibroblast proliferation may inhibit the growth of other cell types. Recent studies have also begun to assign specific roles to different cytokines in embryonic development and cellular differentiation. The growth response to some cytokines may be dependent upon the induction of other cytokines that then directly stimulate proliferation. Some cytokines may also modulate the responses induced by other cytokines by regulating receptor expression and other cellular events. Binding of cytokines to their specific membrane-associated receptors results in activation of signal transduction pathways, many of which involve phosphorylation of specific intercellular proteins. The final biological response is dependent ultimately on the induction and activation of trans-acting factors that regulate transcription by interacting with cis-acting DNA sequences in the target genes. Since many of these signaling and transcriptional events are common to distinct cytokines, many of which have different effects on proliferation in the same cell types, it is not clear how specificity is achieved in regulating the different biological responses.

Animals↗

PubMed1991

Transcription of the muscle regulatory gene Myf4 is regulated by serum components, peptide growth factors and signaling pathways involving G proteins.

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell-surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle-specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.

Adenylate Cyclase Toxin↗

PubMed1991

Interleukin-6 and 12-O-tetradecanoyl phorbol-13-acetate act synergistically in inducing cell-cell separation and migration of human breast carcinoma cells.

Interleukin-6 (IL-6) causes an epithelial to fibroblastoid conversion and an increase in the motility of human ductal breast carcinoma cell lines ZR-75-1 and T-47D. Although IL-6 decreases DNA synthetic activity in these cell lines, the IL-6-induced alterations in cell shape and motility occur independently of inhibition of DNA synthesis per se. Whereas tumor necrosis factor alpha (TNF-alpha) inhibits DNA synthesis in T-47D cells, it does not cause an epithelial-fibroblastoid conversion or other major morphological changes and does not increase cell motility; TNF-alpha rapidly lyses a majority of ZR-75-1 cells. Furthermore, the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine (FUDR) and methotrexate (MTX) also do not cause effects mimicking the action of IL-6 on cell structure and motility. Transforming growth factors alpha and beta 1, acidic and basic fibroblast growth factors, epidermal growth factor, and insulin-like growth factor-1 (TGF-alpha, TGF-beta 1, aFGF, bFGF, EGF, and IGF-1) have little or no effect on breast cancer cell morphology, which serves to exclude the possibility that the IL-6-induced changes are a consequence of induction of these growth factors by IL-6. 12-O-tetradecanoyl phorbol-13-acetate (TPA) but not 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) induces changes in the morphology and associative behavior of ZR-75-1 cells that are similar but not identical to those caused by IL-6. The TPA-induced alterations are not blocked by anti-IL-6 neutralizing antibodies; staurosporine inhibits the TPA-induced cell alterations but not those induced by IL-6. IL-6 and TPA used together have a phenotypic effect that greatly exceeds that of either agent alone and results in extensive cell scattering in less than 1 day. These findings are consistent with the hypothesis that IL-6 and TPA induce similar morphological changes and cell scattering via independent pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Interactions of tumor necrosis factor with local and systemic factors in fetal rat limb bones.

In many tissues the actions of tumor necrosis factor-alpha (TNF) are indirectly mediated through the production of autacoids or other cytokines. To determine the role that these factors might have in the action of TNF on bone resorption, we examined the effects of several selective inhibitors on TNF-stimulated resorption. The cyclooxygenase inhibitor indomethacin did not prevent TNF-stimulated resorption in fetal rat limb bones. Stimulation of resorption by TNF was also unaffected by the platelet activating factor antagonist WEB 2086. A 17.5 kD interleukin receptor antagonist protein, at concentrations that completely blocked the bone-resorbing actions of maximally effective concentrations of interleukin (IL)-1 beta, failed to affect the stimulatory actions of TNF. TNF-stimulated resorption was inhibited by both interferon-gamma and dexamethasone. Dexamethasone inhibited TNF-stimulated resorption more effectively than it inhibited parathyroid hormone (PTH)-stimulated resorption. When bones were treated simultaneously with low concentrations of TNF and PTH, potentiation of the bone-resorbing effects was elicited. These results suggest that TNF stimulates resorption through a pathway different from that by which PTH produces its effects. Transforming growth factor-beta (TGF-beta) enhanced responses to TNF; TGF-beta failed to inhibit the effects of TNF, even in long-term culture or when bones were pretreated with TGF-beta. Synergistic interactions between TNF and several other bone-resorbing factors have now been demonstrated. In contrast to the actions of TNF on certain other functions, the bone-resorbing effects of TNF, as determined in the fetal rat limb bone system, do not seem to be mediated by PAF, IL-1, or prostaglandins.

Animals↗

PubMed1992

A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells.

Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.

Blotting, Northern↗

PubMed1992

Growth factor requirements for DNA synthesis by Leydig cells from the immature rat.

Puberty in the male is dependent upon the elevated production of testosterone by the Leydig cells. LH affects this increase in testosterone output by increasing the total number of Leydig cells in the testis and by stimulating the steroidogenic pathway in these cells. Since Leydig cell proliferation is a prerequisite for the onset of puberty, we have examined the ability of LH and growth factors known to be present in the testis to promote DNA synthesis. Leydig cells were isolated from 21-day-old rats, cultured in serum-free medium for 48 h to become quiescent, and then treated with LH and growth factors for 18 h. [3H]Thymidine incorporation into DNA was assessed over the subsequent 4-h incubation period. Cells in control cultures incorporated low levels of [3H]thymidine into DNA and were stimulated after treatment with LH (100 ng/ml). Insulin/insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), previously localized in Leydig cells by immunohistochemistry, also stimulated [2H]thymidine incorporation into DNA. The responses of the Leydig cells to maximum levels of insulin and TGF-alpha were dependent on the cell density. Insulin and TGF-alpha alone and in combination increased the number of cells labeled with [3H]thymidine, as assessed by autoradiography. TGF-beta, known to be secreted by Sertoli cells, also stimulated DNA synthesis under basal conditions, but the maximum response was significantly lower than that achieved in the presence of TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Pathways of fibrin turnover of human pleural mesothelial cells in vitro.

The mesothelium contains both procoagulant and fibrinolytic activities. An imbalance between these activities could account for the abnormal fibrin turnover and pleural fibrin deposition that is characteristic of pleural inflammation. Procoagulant activity of human pleural mesothelial cells (HPMC) is in part due to tissue factor, and the prothrombinase complex can also assemble at the HPMC surface. HPMC express tissue plasminogen activator (tPA) but no detectable fibrinolytic activity in a fibrin plate assay. Inhibition of HPMC fibrinolytic activity is due, in part, to elaboration of plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) as well as antiplasmins. Synthesis of PAI-1 and PAI-2 is inhibited by actinomycin D and cyclohexamide. HPMC PAI-1 is increased by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), as is tPA release, while PAI-1 mRNA is unchanged and tPA mRNA is increased. PAI-2 release is induced by TNF-alpha and TGF-beta. Because they are a rich source of PAI-1 and PAI-2, HPMC may contribute to the high levels of these inhibitors in pleural exudates. Stimulation of HPMC by TNF-alpha or TGF-beta in vitro did not alter HPMC procoagulant activity nor the balance of elevated PAI and antiplasmins relative to PA, changes that collectively favor formation and persistence of pericellular fibrin.

Base Sequence↗

PubMed1992

Renal extracellular matrix accumulation in acute puromycin aminonucleoside nephrosis in rats.

Progressive renal fibrosis is considered to be the final common pathway leading to chronic renal insufficiency. In this study, the authors examined some of the cellular and molecular mechanisms regulating the renal accumulation of extracellular matrix (ECM) proteins using rats with puromycin amino-nucleoside (PAN) nephrosis as an acute model system. Puromycin aminonucleoside rats developed reversible nephrotic syndrome accompanied by an interstitial infiltrate of monocytes. The number of interstitial fibroblasts expressing ST4 antigen did not increase. During the first 4 days, steady-state mRNA levels for all genes examined remained at or below control levels. At 1 week, nephrotic syndrome and interstitial inflammation were established, and a period of renal cell proliferation occurred, identified by increased histone mRNA levels and localized by tritiated thymine autoradiography to tubular epithelial cells and occasional interstitial cells. Transforming growth factor-beta (TGF-beta) steady-state mRNA levels were increased eightfold, but returned to control levels by 3 weeks. At week 1, there was a 10- to 20-fold increase in kidney steady-state mRNA levels for genes encoding interstitial matrix proteins collagen I and fibronectin and basement membrane collagen IV. By in situ hybridization, alpha 1(I) procollagen mRNA was localized to interstitial cells. Immunofluorescence microscopy demonstrated focal accumulation of ECM proteins in the tubulointerstitial compartment at 2 and 3 weeks, but by 6 weeks, kidney immunohistology was normal again. Steady-state mRNA levels for the matrix degrading metalloproteinase stromelysin remained at control values, whereas the levels for interstitial collagenase were normal at week 1 and increased twofold to threefold at 2 and 3 weeks. Steady-state mRNA levels for the tissue inhibitor of metalloproteinases (TIMP) increased fivefold at 1 week and returned to baseline values over the next 2 weeks. The results of this study suggest that tubulointerstitial ECM accumulation occurs in rats with acute PAN nephrosis because of the activation of genes encoding several matrix proteins and inhibition of matrix degradation mediated by TIMP. These events are reversed during the phase of recovery from nephrotic syndrome. Increased mRNA levels for TGF-beta, possibly originating from inflammatory interstitial monocytes, are likely to be one of the mediators of the molecular events observed.

Animals↗

PubMed1992

Vascular smooth muscle growth in genetic hypertension: evidence for multiple abnormalities in growth regulatory pathways.

OBJECTIVE: To gain insight into the mechanisms which contribute to the development of vascular hypertrophy in the spontaneously hypertensive rat (SHR). DESIGN: These experiments were performed under conditions which most closely mimic the growth of smooth muscle in blood vessels, i.e. once cell-cell contact has been achieved. METHODS: A comparison of the growth characteristics (growth rates and cell density at quiescence) of vascular smooth muscle cells (VSMC) from SHR and normotensive Wistar-Kyoto (WKY) rats. RESULTS: In the presence of foetal calf serum (1, 2.5, 5 and 10%), early passaged VSMC from SHR exhibited higher growth rates and reached higher densities at quiescence than VSMC from WKY rats. Accelerated growth rates could not be attributed to differences in cell-cell interactions. Also, growth rates and cell density at quiescence appear to be regulated by distinct mechanisms. Transforming growth factor-beta 1 (TGF-beta 1) caused an inhibition of serum-stimulated proliferation of confluent VSMC from WKY rats. In contrast, TGF-beta 1 had little, if any, inhibitory action upon the growth of VSMC from SHR. Scatchard analysis of 125I-TGF-beta 1 binding to VSMC from both strains yielded a single class of high affinity binding sites. CONCLUSIONS: VSMC from SHR exhibit enhanced proliferation, attain a higher cell density at quiescence and are less susceptible to growth inhibition by TGF-beta 1 than VSMC from WKY rats. All these characteristics of SHR VSMC may contribute to the development of vascular hypertrophy in this strain.

Animals↗

PubMed1992

Increased expression of genes from growth factor signaling pathways in glioblastoma cell lines.

The concept of autocrine stimulation of cell proliferation postulates growth autonomy by acquisition of the ability to produce and respond to growth factors. Overproduction of several growth factors in a variety of human tumors and cell lines derived from these tumors has been reported. We have screened several cell lines derived from glioblastomas for anomalies in the expression of genes encoding transforming growth factor alpha (TGF-alpha), TGF-beta, basic fibroblast growth factor (bFGF) and its high-affinity receptor, flg. Compared with normal human brain tissue, we observed a generalized elevation in the levels of expression of these genes in glioblastoma cell lines and an SV40-transformed human astroglial cell line. Overexpression of these genes does not appear to be merely a reflection of the proliferative state of transformed cells since some other human tumor cell lines, when analysed for the expression of TGF-beta and bFGF, did not show a significant increase in these transcripts. The specificity of the elevated transcription of TGF-alpha, TGF-beta, bFGF and flg in glioblastoma cell lines is further suggested by the fact that the transcription of the proto-oncogene c-erbB2, which is overproduced in breast tumor cell lines, was not elevated in glioblastoma cell lines. Increased expression of growth factors, which are potent mitogens and angiogens, and/or their receptors may have critical roles in autonomous proliferation as well as neovascularization of glioblastomas.

Fibroblast Growth Factor 2↗

PubMed1992

Type beta transforming growth factors promote interleukin-3 (IL-3)-dependent differentiation of human basophils but inhibit IL-3-dependent differentiation of human eosinophils.

Basophils and eosinophils share a common differentiation pathway. Factors regulating terminal commitment toward one cell type, however, have so far not been defined. Interleukin-3 (IL-3) is a potent differentiation factor for both human eosinophils and basophils. In the present study, the effects of various recombinant human (rh) growth regulators on IL-3-dependent growth of eosinophils and basophils were studied in a bone marrow (BM) suspension culture system (normal donors, n = 13). We found that type beta transforming growth factors (TGFs) lead to a significant increase in the absolute numbers of basophils in BM cultures grown in the presence of IL-3 (day 14 of culture; IL-3: 133 +/- 20 v IL-3 + TGF-beta 1: 231 +/- 28 x 10(3)/mL [P less than .01]) and to an increase in the total histamine values (IL-3: 72.6 +/- 22.2 v IL-3 + TGF-beta 1: 142.9 +/- 37.3 ng/mL [P less than .015]) compared with rhIL-3 alone. In contrast, type beta TGFs were found to inhibit the IL-3-dependent growth of eosinophils (IL-3: 170.4 +/- 37.2 v IL-3 + TGF-beta 1: 16.7 +/- 5.2 x 10(3)/mL [P less than .01]) and formation of eosinophil cationic protein in the same culture system. The effect of TGF-beta 1 (and TGF-beta 2) on IL-3-dependent differentiation of basophils and eosinophils was dose- and time-dependent (maximum effects observed with 1 to 10 ng/mL of rhTGF-beta 1 or TGF-beta 2) and could be neutralized by an antibody specific for TGF-beta 1. In contrast to the TGFs, interferon-alpha (IFN-alpha) and IFN-gamma were found to downregulate IL-3-dependent formation of both basophils (IL-3: 167 +/- 33 v IL-3 + IFN-alpha: 67 +/- 25 v IL-3 + IFN-gamma: 65 +/- 33 x 10(3)/mL [P less than .01]) and eosinophils (IL-3: 239 +/- 5 v IL-3 + IFN-alpha: 81 +/- 4 v IL-3 + IFN-gamma: 67 +/- 17 x 10(3)/mL [P less than .05]) in our culture system. Type beta TGFs as well as the IFNs failed to directly induce differentiation of human basophils or eosinophils in the absence of other growth factors. Together, these results show that type beta TGFs and IFNs are potent regulators of cytokine-dependent growth and differentiation of human allergic effector cells.

Antibodies, Monoclonal↗

PubMed1992

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.

Amino Acid Sequence↗

PubMed1992

Anti-immunoglobulin treatment of murine B-cell lymphomas induces active transforming growth factor beta but pRB hypophosphorylation is transforming growth factor beta independent.

Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. When WEHI-231 lymphoma cells were selected for growth in TGF-beta, the majority of the TGF-beta-resistant clones remained sensitive to anti-Ig-mediated growth inhibition. In these clones, the retinoblastoma gene product was found to be in the underphosphorylated form after 24-h treatment with anti-Ig, but not with TGF-beta. These data show that anti-Ig treatment of murine B-cell lymphomas stimulates the production of active TGF-beta but that a TGF-beta-independent pathway may be responsible for the pRB underphosphorylation and cell cycle blockade.

Animals↗

PubMed1991

Transforming growth factor-beta-induced inhibition of T cell function. Susceptibility difference in T cells of various phenotypes and functions and its relevance to immunosuppression in the tumor-bearing state.

The present study investigates the nature of humoral component(s) generated in tumor-bearing hosts to induce immune dysfunction of T cells. Cell-free ascitic fluid and culture supernatant (SN) were obtained from the ascites and cultures allowing MH134 hepatoma cells to grow. These ascites and SN samples were tested for their abilities to influence the generation of CTL responses to TNP and alloantigens. The generation of the anti-TNP CTL responses that require self H-2-restricted CD4+ Th cells was markedly suppressed by addition of the ascites or SN under conditions in which these samples did not inhibit anti-allo CTL responses capable of using alternate pathways of allo-restricted CD4+ and CD8+ Th. The activation of CD8+ CTL precursors and CTL activity were also resistant to the ascites or SN. The ascites- or SN-induced suppressive effect to which CD4+ Th were most susceptible was found to be mediated by transforming growth factor-beta (TGF-beta) activity, because: 1) the TGF-beta activity was detected in the MH134 ascites and culture SN; 2) the suppression of CD4+ Th function required for anti-TNP CTL responses was almost completely prevented by addition of anti-TGF-beta antibody to cultures and; 3) rTGF-beta also induced similar patterns of immunosuppression to those observed by ascites or SN. These results indicate that TGF-beta produced by tumor cells induces deleterious effects on T cell, especially on the CD4+ Th subset, and provide an explanation for the molecular mechanism underlying the previously observed CD4+ Th-selective suppression in the tumor-bearing state.

Animals↗