Differential regulation of mouse B cell development by transforming growth factor beta1

Transforming growth factor beta (TGFbeta) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFbeta1-/- mice. To evaluate TGFbeta responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7) +/- TGFbeta. Picomolar doses of TGFbeta1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFbeta1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFbeta1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.     

Immunohistochemical analysis of hepatocyte growth factor in human coronary atherectomy specimens: comparison with transforming growth factor beta isoforms

The expression and localization of hepatocyte growth factor/scatter factor (HGF/SF) were examined immunohistochemically in 59 human coronary artery lesions retrieved by directional coronary atherectomy and compared with the localization of transforming growth factor beta isoforms (TGF-β1, -β2, and -β3). In 21 of the 59 specimens (35.6%) HGF-like immunoreactivity (HGF-IR) was revealed. The HGF immunopositivity rate of 45% (14/31) in thrombotic tissue was significantly (P < 0.05) higher than the rates of 7.3% (4/55), 7.1% (3/42), and 0% (0/14) in fibrous tissue, neointimal hyperplasia and atheromatous gruel, respectively. Immunoreactivity for HGF was much weaker than that for TGF-β isoforms in these components except in thrombotic tissue. These cells exhibiting strong HGF-IR were inflammatory cells such as monocytes/macrophages in thrombotic tissue, in tissue lesions adjacent to a thrombus, and outside the capillary walls in a portion of the neovascularized lesions. Smooth muscle cells (SMCs) hardly demonstrated HGF-IR. In contrast, in control coronary arteries obtained at autopsy, the HGF-IR was strongly expressed in SMCs. These findings suggest that HGF produced by macrophages play a part in the process of coronary plaque formation attributable to thrombus in man.     

Neutralizing antibodies against transforming growth factor beta potentiate the proliferation of Ki-1 positive lymphoma cells. Further evidence for negative autocrine regulation by transforming growth factor beta.

Activated lymphocytes and malignant lymphoma cells derived from them (Ki-1 positive lymphoma cells) share similar mechanisms of proliferation. To further examine the inhibitory role of endogenous transforming growth factor beta (TGF beta) in Ki-1 positive lymphoma cells, the authors studied anti-TGF beta antibodies and measured their effect on proliferation. A monoclonal antibody (T1A5) prepared against a unique antigenic epitope of high molecular weight Hodgkin's TGF beta and a polyclonal rabbit antibody prepared against highly purified 25,000 D porcine platelet TGF beta 1 were used. Both antibodies are shown here to inhibit the biological activity of Hodgkin's TGF beta and to crossreact with their respective antigens in immunoblotting. DNA synthesis by Ki-1 lymphoma cells was increased 138-fold by anti-TGF beta 1 antibody and 262-fold by anti-Hodgkin's TGF beta. Exogenous TGF beta 1 suppression was completely reversed by anti-TGF beta 1 antibody and IL-2-induced proliferation was markedly potentiated (41 fold). L-428 Reed-Sternberg cells secrete physiologically active TGF beta but have fewer than 500 TGF beta receptor sites per cell; no significant proliferative response was measured for either anti-TGF beta 1 or anti-Hodgkin's TGF beta. These results show the suppressive effect of exogenous TGF beta 1 on indolent Ki-1 lymphoma cells and suggest that the endogenous secretion of high molecular weight physiologically active TGF beta is important in maintaining the indolent nature of this low-grade Ki-1 positive lymphoma.     

Interleukin 7 (murine pre-B cell growth factor/lymphopoietin 1) stimulates thymocyte growth: regulation by transforming growth factor beta

The effects of interleukin 7 (IL7) previously known as murine pre-B cell growth factor/lymphopoietin 1 on the growth of murine thymocytes was investigated. In the presence of a suboptimal dose of phytohemagglutinin, IL7 induced a dose-dependent increase in thymocyte proliferation which was comparable to that induced by IL1. Additionally IL7 was shown to synergize with a suboptimal dose of IL1 to enhance thymocyte proliferation. Thymocyte proliferation induced by IL7, like that induced by IL1, was inhibited when either recombinant transforming growth factor (TGF) beta 1 or beta 2 were added at the initiation of culture. Interestingly, IL7-driven thymocyte proliferation was considerably less susceptible to inhibition by TGF-beta 1 or TGF-beta 2 than that induced by IL1. Taken together these results suggest IL7 may activate distinct populations of thymocytes and/or act through a pathway distinct from that utilized by IL1.     

Bioactivation of latent transforming growth factor beta1 by Mycobacterium tuberculosis in human mononuclear phagocytes

Biologically active transforming growth factor beta 1 (TGFbeta1) has been identified at sites of Mycobacterium tuberculosis (MTB) infection in the lung; however, the underlying mechanism(s) for its activation is not clear. Here using an enzyme-linked immunospot assay for TGFbeta1, we show that human blood monocytes (MN) and alveolar macrophages (AM) produce bioactive TGFbeta1 upon stimulation by MTB. However, only MTB-stimulated MN increased TGFbeta1 production on a per cell basis. The frequency of TGFbeta1-producing MN was reduced by an inhibitor of plasmin, bdellin, indicating a role for plasmin pathways in the bioactivation of cytokine. The expression of urokinase plasminogen activator receptor (uPAR) mRNA and both surface and soluble uPAR (CD87) was increased in MTB-activated MN. However, antibody neutralization of uPAR suppressed bioactive TGFbeta1 in MN alone. Thus, the more immature MN, which are continuously recruited to the lung during tuberculosis (TB), have a higher capacity to bioactivate TGFbeta1 by expression of components of the plasmin pathway. Excess production and bioactivation of TGFbeta1 at sites of MTB infection may undermine host immune responses during TB.     

人角质形成细胞Notch信号通路与转化生长因子β调控受体蛋白的作用

背景：在皮肤中受体蛋白酪氨酸磷酸酶kappa的调控至关重要,而转化生长因子β似乎是其调控的上游因子,既然Notch信号和转化生长因子β信号通道如此相关,那么Notch是不是也参加了转化生长因子β信号对受体蛋白酪氨酸磷酸酶kappa的调控呢？ 目的：探讨Notch信号通道在人角质形成细胞中对转化生长因子β调控受体蛋白酪氨酸磷酸酶kappa的作用的影响。 方法：在分别用Jagged-1激活和用Γ-分泌酶抑制剂抑制Notch信号通道后,加入转化生长因子β,同时设立对照组,用Real-time PCR测试人角质形成细胞中受体蛋白酪氨酸磷酸酶kappa mRNA表达量。 结果与结论：覆盖率为40%的角质形成细胞在加入了转化生长因子β后,受体蛋白酪氨酸磷酸酶kappa mRNA量在各时间点均高于对照组。在用Jagged-1激活Notch通道的角质形成细胞中,单独加入Jagged-1、转化生长因子β及两者都加入时均高于对照组(P < 0.05,P < 0.01)。在用γ-分泌酶抑制剂抑制Notch通道的角质形成细胞中,只加入转化生长因子β显著高于对照组(P < 0.01),只加入γ-分泌酶抑制剂和两者均加入时与对照组比较,差异无显著性意义(P > 0.05)。说明加入转化生长因子β导致角质形成细胞中受体蛋白酪氨酸磷酸酶kappa表达增加,而分别对Notch信号进行激活和抑制后发现,受体蛋白酪氨酸磷酸酶kappa信号分别显著增加和显著被抑制。所以在转化生长因子β升高受体蛋白酪氨酸磷酸酶kappa表达过程中Notch信号通道是非常重要且不可或缺的。     

Effect of transforming growth factor beta 1 on neonatal rat latent transforming growth factor beta binding protein 2 in cardiomyocytes

目的 探讨转化生长因子β1 (TGF-β1)在诱导心肌细胞表达转化生长因子结合蛋白2(LTBP2)中的作用及信号传导通路.方法 培养乳鼠心肌细胞;实时定量聚合酶链式反应、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制.结果 LTBP2基因表达随着TGF-β1浓度增加(0、2、5及10 μg/L)而明显升高,在5μg/L时刺激最强(P＜0.05);5μg/L的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势(P＜0.05或P＜0.01);免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达.信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达.结论TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达.     

转化生长因子β1对乳鼠心肌细胞转化生长因子结合蛋白2表达的影响

 目的：探讨转化生长因子β1（TGF-β1）在诱导心肌细胞表达转化生长因子结合蛋白2（LTBP2）中的作用及信号传导通路。 方法：培养乳鼠心肌细胞;实时定量聚合酶链式反应（Real-time PCR）、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制。 结果：LTBP2基因表达随着TGF-β1浓度增加（0、2、5、10 ng/mL）而明显升高,在5 ng/mL时刺激最强（P < 0.05）;5 ng/mL的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势（P < 0.05或P<0.01）;免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达。信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达。 结论：TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达。     

Inhibition of myofibroblast apoptosis by transforming growth factor beta(1)

Fibroblast differentiation to the myofibroblast phenotype is associated with alpha-smooth-muscle actin (alpha-SMA) expression and regulated by cytokines. Among these, transforming growth factor (TGF)-beta(1) and interleukin (IL)-1beta can stimulate and inhibit myofibroblast differentiation, respectively. IL-1beta inhibits alpha-SMA expression by inducing apoptosis selectively in myofibroblasts via induction of nitric oxide synthase (inducible nitric oxide synthase [iNOS]). Because TGF-beta is known to inhibit iNOS expression, this study was undertaken to see if this cytokine can protect against IL-1beta-induced myofibroblast apoptosis. Rat lung fibroblasts were treated with IL-1beta and/or TGF-beta(1) and examined for expression of alpha-SMA, iNOS, and the apoptotic regulatory proteins bax and bcl-2. The results show that TGF-beta(1) caused a virtually complete suppression of IL-1beta-induced iNOS expression while preventing the decline in alpha-SMA expression or the myofibroblast subpopulation. TGF-beta(1) treatment also completely suppressed the IL-1beta-induced apoptosis in myofibroblasts. IL-1beta-induced apoptosis was associated with a significant decline in expression of the antiapoptotic protein bcl-2, which was prevented by concomitant TGF-beta(1) treatment. The level of the proapoptotic protein bax, however, was not significantly altered by either cytokine. These data suggest that TGF-beta(1) inhibits IL-1beta-induced apoptosis in myofibroblasts by at least two mechanisms, namely, the suppression of iNOS expression and the prevention of a decline in bcl-2 expression. Thus, TGF-beta(1) may be additionally important in fibrosis by virtue of this novel ability to promote myofibroblast survival by preventing the myofibroblast from undergoing apoptosis.     

Fibroblast growth factor 2 and transforming growth factor beta1 synergism in human bronchial smooth muscle cell proliferation

Bronchial smooth muscle cell (BSMC) hyperplasia is a typical feature of airway remodeling and contributes to airway obstruction and hyperresponsiveness in asthma. Fibroblast growth factor 2 (FGF-2) and transforming growth factor beta1 (TGF-beta1) are sequentially upregulated in asthmatic airways after allergic challenge. Whereas FGF-2 induces BSMC proliferation, the mitogenic effect of TGF-beta1 remains controversial, and the effect of sequential FGF-2 and TGF-beta1 co-stimulation on BSMC proliferation is unknown. This study aimed to assess the individual and sequential cooperative effects of FGF-2 and TGF-beta1 on human BSMC proliferation and define the underlying mechanisms. Mitogenic response was measured using crystal violet staining and [3H]-thymidine incorporation. Steady-state mRNA and protein levels were measured by semiquantitative RT-PCR, Western blot, and ELISA, respectively. TGF-beta1 (0.1-20 ng/ml) alone had no effect on BSMC proliferation, but increased the proliferative effect of FGF-2 (2 ng/ml) in a concentration-dependent manner (up to 6-fold). Two distinct platelet-derived growth factor receptor (PDGFR) inhibitors, AG1296 and Inhibitor III, as well as a neutralizing Ab against PDGFRalpha, partially blocked the synergism between these two growth factors. In this regard, TGF-beta1 increased PDGF-A and PDGF-C mRNA expression as well as PDGF-AA protein expression. Moreover, FGF-2 pretreatment increased the mRNA and protein expression of PDGFRalpha and the proliferative effect of exogenous PDGF-AA (140%). Our data suggest that FGF-2 and TGF-beta1 synergize in BSMC proliferation and that this synergism is partially mediated by a PDGF loop, where FGF-2 and TGF-beta1 upregulate the receptor (PDGFRalpha) and the ligands (PDGF-AA and PDGF-CC), respectively. This powerful synergistic effect may thus contribute to the hyperplastic phenotype of BSMC in remodeled asthmatic airways.     

转化生长因子β1对横纹肌肉瘤RD细胞系的生长调节及机制探讨

目的 研究转化生长因子(TGF)-β1对横纹肌肉瘤RD细胞系的生长调节及作用机制。方法^3H-thymidine掺入实验、四甲基偶氮唑盐(MTT)实验和生长曲线检测经TGF-β1处理不同时间的RD细胞生长活力的变化;应用流式细胞术榆测RD细胞周期的改变;激光扫描共聚焦显微镜观察细胞周期抑制蛋白p15、p21和p27在RD中分布的变化;逆转录一聚合酶链反应(RT-PCR)和Western bolt检测RD细胞中细胞周期抑制蛋白P15、p21、p27mRNA和蛋白水平的变化：结果TGF-β1处理RD细胞后,其牛长活力明显降低,并出现G1期停滞。p21,p27在mRNA和蛋白水平表达上升,且p21南胞核表达改变为胞核胞质内均有表达。p15在mRNA和蛋白水平上均无明显改变。结论 TGF-β1对RD细胞具有生长抑制作用,促使细胞G1期停滞。TGF-β1可在mRNA和蛋白水平上调RD细胞中p21、p27的表达。TGF-β1可能通过上调p2和p27而非p15抑制RD细胞生长。     

Differential effect of transforming growth factor beta on the synthesis of Th1- and Th2-like lymphokines by human T lymphocytes.

(TGF)-beta is a pluripotent cytokine exerting differential effects on distinct components of the immune response. The present report, based on lymphokine determination in culture supernatants and Northern blot analysis of lymphokine mRNA, demonstrates that TGF-beta 2 markedly inhibits interleukin (IL)-4 and IL-5 synthesis by polyclonally activated human T cells in the absence of any significant effect on (IFN)-gamma, lymphotoxin or IL-2, suggesting a modulatory effect of TGF-beta 2 on the interferon Th1/Th2) balance of immune responses. The inhibitory effect of TGF-beta on IFN-gamma production by unfractionated peripheral blood mononuclear cells is likely to reflect the blunting of natural killer cell activation by TGF-beta.     

转化生长因子β1在兔房水中的浓度变化

背景：转化生长因子β1可参与角膜损伤后的修复。 目的：观察转化生长因子β1滴眼液滴眼后房水中的浓度变化规律。 方法：将新西兰大白兔随机分为5组,分别给予PBS和质量浓度为0.5,1.0,2.0,4.0 mg/L的转化生长因子β1滴眼液滴右眼。 结果与结论：通过裂隙灯观察兔角膜和结膜结构,各组兔眼均无结膜分泌物、球结膜充血、角膜水肿增厚、角膜后沉着物、前房炎性反应及晶状体混浊改变。ELISA检测结果显示,与PBS组比较,质量浓度2.0和4.0 mg/L转化生长因子β1滴眼液能有效提高兔眼房水中转化生长因子β1的质量浓度(P < 0.01),角膜穿透性良好,在房水中可以达到有效的治疗浓度。     

Retention of transforming growth factor beta1 using functionalized dextran-based hydrogels

Functionalized dextrans (FD) are anionic water-soluble polymers bearing carboxylate, benzylamide and sulfate groups, which exhibit binding capacity to transforming growth factor-beta1 (TGF-beta1). In this paper, we have investigated the ability of dextran-based hydrogels containing FD, to bind and release recombinant human TGF-beta1. Hydrogels were prepared by chemical crosslink native dextran and FD with sodium trimetaphosphate in 1m NaOH at 50 degrees C. A wide range of hydrogels were prepared as particles ranging of 1-1.6mm of diameter and characterized with various amounts of FD and with different crosslinker feeding ratios (CFR). Dried particles were soaked with recombinant human transforming growth factor-beta1 (rhTGF-beta1) to determine their capacity to deliver the growth factor. Results indicated that the in vitro kinetics releases of rhTGF-beta1 were related to FD and CFR. Retention capacity of rhTGF-beta1 increases with an increase of negative charges of the matrices brought by both phosphate linkages and FD as demonstrated by an additional release of growth factor in high ionic strength solution. Highly crosslinked hydrogels that contained the highest amount of FD (18% (w/w)) retained up to 88% of rhTGF-beta1. Bioactivity of released growth factor was confirmed in a cell assay. These functionalized hydrogels may have important uses for the stabilization and the protection of rhTGF-beta1 as entrapment systems and could be applied to other proteins of clinical interest.     

In vitro human osteoblast and extracellular matrix changes after transforming growth factor beta 1 treatment

AIMS: Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia. METHODS: In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined. RESULTS: Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (p < or = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities. CONCLUSION: TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.     

Regulation of anti-immunoglobulin-induced B lymphoma growth arrest by transforming growth factor beta 1 and dexamethasone.

Exposure of the murine B lymphoma cell line WEHI-231 to anti-immunoglobulin M (anti-IgM) antibodies results in growth arrest in the G1 phase of the cell cycle followed by programmed cell death. This response may be analogous to the clonal deletion of immature B cells that occurs when the membrane IgM on these cells is engaged by self-antigens or by anti-IgM antibodies. Thus the WEHI-231 cell line has been a useful in vitro system for identifying factors that modulate anti-Ig-induced growth inhibition and/or clonal deletion. For example, both antigen-induced tolerance induction in immature B cells and anti-Ig-induced growth arrest of WEHI-231 cells are prevented by bacterial lipopolysaccharide or by the products of activated T helper cells. Since negative signaling by membrane Ig may also be regulated by additional factors, we asked whether other cytokines or hormones would regulate the growth of WEHI-231 cells or its response to anti-IgM antibodies. We show here that two compounds that are generally immunosuppressive, transforming growth factor beta 1 (TGF-beta 1) and the synthetic corticosteroid, dexamethasone, blocked the ability of lipopolysaccharide and T cell-derived lymphokines to protect WEHI-231 cells from anti-IgM-induced growth arrest. In addition, TGF-beta 1 and dexamethasone slightly inhibited the growth of WEHI-231 cells by themselves and also potentiated the growth inhibitory effects of anti-IgM antibodies. Thus for WEHI-231 cells, TGF-beta 1 and dexamethasone are inhibitory factors which favor growth arrest.     

Continuous transforming growth factor beta1 secretion by cell-mediated gene therapy maintains chondrocyte redifferentiation

One of the most important factors in the production of cartilage is transforming growth factor beta1 (TGF-beta1). To obtain sustained release of TGF-beta1, a cell-mediated gene therapy technique was introduced. We infected chondrocytes with a retroviral vector carrying the TGF-beta1 gene. The single clone derivative showed sustained TGF-beta1 secretion. It also showed constitutive type II collagen expression. Whereas the TGF-beta1 protein itself is unable to induce formation of cartilage in vivo, human chondrocytes engineered to express a retroviral vector encoding TGF-beta1 showed cartilage formation in vivo when cells were injected into nude mice intradermally. These data suggest that cell-mediated gene therapy using TGF-beta1 as a transgene would be a promising treatment for osteoarthritis.