Transforming Growth Factor-β Secretion Research Articles

Mast Cells: A Pivotal Role in Pulmonary Fibrosis

Pulmonary fibrosis is characterized by an inflammatory response that includes macrophages, neutrophils, lymphocytes, and mast cells. The purpose of this study was to evaluate whether mast cells play a role in initiating pulmonary fibrosis. Pulmonary fibrosis was induced with bleomycin in mast-cell-deficient WBB6F1-W/W(v) (MCD) mice and their congenic controls (WBB6F1-(+)/(+)). Mast cell deficiency protected against bleomycin-induced pulmonary fibrosis, but protection was reversed with the re-introduction of mast cells to the lungs of MCD mice. Two mast cell mediators were identified as fibrogenic: histamine and renin, via angiotensin (ANG II). Both human and rat lung fibroblasts express the histamine H1 and ANG II AT1 receptor subtypes and when activated, they promote proliferation, transforming growth factor β1 secretion, and collagen synthesis. Mast cells appear to be critical to pulmonary fibrosis. Therapeutic blockade of mast cell degranulation and/or histamine and ANG II receptors should attenuate pulmonary fibrosis.

Activation of inflammatory cells and cytokines by peptide epitopes in vitro: a simple in-vitro screening assay for prioritizing them for in-vivo studies

Antigen-specific immune modulation is an attractive approach to atherosclerosis treatment. The aim of this study was to develop an in-vitro assay to screen peptide molecules for their inflammatory propensity. Human dendritic cells derived from CD14(+) monocytes were activated using peptides derived from apolipoprotein B100 (ApoB), heat shock protein 60 (HSP60) and complement cascade (peptide A) in vitro, and used for priming autologous T cells. Proliferation of T cells, their differentiation to regulatory cells (Treg) and their cytokine profile were studied. The efficacy of the peptides in preventing atherosclerosis was studied in ApoB(tm2Sgy)/Ldlr(tm1Her/J) knockout mice. ApoB and HSP60 peptides induced T-cell proliferation and expansion of regulatory T cells with interleukin-10 and transforming growth factor-β secretion. In comparison, peptide A was a poor stimulator of T cells and was found to induce tumor necrosis factor-α secretion by activated T cells. ApoB and HSP60 peptides were found to reduce early atherosclerotic lesion formation in mice by 32.1 and 33.5%, respectively, while the reduction with peptide A was 5.7%. Thus the in-vitro assay shows an apparent correlation with in-vivo activity and can be developed as a screening assay to prioritize the candidate molecules for animal efficacy testing.

Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells

To investigate cytokine production and cell surface phenotypes of dendritic cells (DC) in the presence of epithelial cells stimulated by probiotics. Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or inverted systems and were stimulated with heat-killed probiotic bacteria, Bifidobacterium lactis AD011 (BL), Bifidobacterium bifidum BGN4 (BB), Lactobacillus casei IBS041 (LC), and Lactobacillus acidophilus AD031 (LA), for 12 h. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent assay and phenotypic analysis of DC was investigated by flow cytometry. BB and LC in single-cultured DC increased the expression of I-Ad, CD86 and CD40 (I-Ad, 18.51 vs 30.88, 46.11; CD86, 62.74 vs 92.7, 104.12; CD40, 0.67 vs 6.39, 3.37, P < 0.05). All of the experimental probiotics increased the production of inflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α. However, in the normal co-culture systems, LC and LA decreased the expression of I-A(d) (39.46 vs 30.32, 33.26, P < 0.05), and none of the experimental probiotics increased the levels of IL-6 or TNF-α. In the inverted co-culture systems, LC decreased the expression of CD40 (1.36 vs -2.27, P < 0.05), and all of the experimental probiotics decreased the levels of IL-6. In addition, BL increased the production of IL-10 (103.8 vs 166.0, P < 0.05) and LC and LA increased transforming growth factor-β secretion (235.9 vs 618.9, 607.6, P < 0.05). These results suggest that specific probiotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.

Mesenchymal Stem Cells Negatively Regulate Dendritic Lineage Commitment of Umbilical-Cord-Blood-Derived Hematopoietic Stem Cells: An Unappreciated Mechanism as Immunomodulators

Due to their immunomodulatory functions, mesenchymal stem cells (MSCs) have great potential for clinical applications to prevent rejection in organ transplantation and to prevent graft-versus-host disease in hematopoietic stem cell (HSC) transplantation. Since dendritic cells (DCs) play an important role in modulating diverse T cell responses, including rejection and graft-versus-host disease, the goal of this study was to investigate whether MSCs modulate DC differentiation from HSCs and if this effect could be one of the mechanisms for MSCs' immune-modulating functions. Our results demonstrate that differentiation of HSCs into mature DCs is inhibited in the presence of MSCs. Similar frequency of dendritic precursors in the cultures, either with or without MSCs, suggests that the inhibition of MSCs on the differentiation of mature DCs from HSCs could be due to the arresting of maturation at the dendritic precursor step. Reduced levels of cyclic AMP, adenosine 3',5'-cyclic monophosphate (cAMP) and beta-catenin in DC-like cells from the cocultures are detected, suggesting that induction of apoptosis and inhibition of differentiation could be the basis for the inhibition of mature DCs from HSCs by MSCs. Further, our results demonstrate that DCs derived from HSCs in the presence of MSCs are functionally impaired, especially for those after direct contact with MSCs. To investigate the basis of functional impairment, our data show downregulated tumor necrosis factor-alpha and transforming growth factor-beta1 secretion and upregulated interleukin-6 (IL6) and IL1beta secretion in the cultures with MSCs. Together, MSCs can inhibit differentiation of mature DCs from HSCs by arresting them at the precursor stage and induce their apoptosis. Further, HSC-derived DCs in the presence of MSCs are functionally impaired, which could be partly due to the upregulation of IL6 secretion.

Transforming growth factor β1 secretion and receptor expression are dysregulated with the progression of chronic venous insufficiency

Transforming growth factor β1 secretion and receptor expression are dysregulated with the progression of chronic venous insufficiency

Intratumoral Induction of CD103 Triggers Tumor-Specific CTL Function and CCR5-Dependent T-Cell Retention

We have reported previously that the interaction of alpha(E)(CD103)beta(7) integrin, expressed on a CD8(+) tumor-infiltrating lymphocyte (TIL) clone but not on a peripheral blood lymphocyte (PBL) counterpart, with the epithelial marker E-cadherin on human lung tumor cells plays a crucial role in T-cell receptor-mediated cytotoxicity. We show here that both TIL and PBL clones are able to migrate toward autologous tumor cells and that chemokine receptor CCR5 is involved in this process. Adoptive transfer of the PBL clone in the cognate tumor engrafted in nonobese diabetic/severe combined immunodeficient mice and subsequent coengagement of T-cell receptor and transforming growth factor-beta1 receptor triggers CD103 expression on T-cell surface resulting in strong potentiation of antitumor lytic function. Moreover, interaction of alpha(E)beta(7) integrin with E-cadherin, but not lymphocyte function-associated antigen-1 with intercellular adhesion molecule-1, promotes CCR5 recruitment at the immunologic synapse formed between TIL and tumor cells, leading to inhibition of T-cell sensitivity to CCL5 chemotactic gradient. These results provide evidence for a role of tumor microenvironment, namely MHC class I-restricted antigen presentation and transforming growth factor-beta1 secretion, in regulating the effector phase of tumor-specific CTL response. They also suggest a unique role of CD103 in T-cell retention at the tumor site by a CCR5-dependent mechanism.

The involvement of transforming growth factor-β1 secretion in Urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-β1 (TGF-β1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-β1, and the role of U II receptor UT in this process. The functional role of TGF-β1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-β1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-β1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-β1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-β1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-β1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.

High glucose-induced oxidative stress inhibits Na+/glucose cotransporter activity in renal proximal tubule cells

Oxidative stress plays an important role in the pathogenesis of renal diseases such as diabetic nephropathy. The metabolism of excessive intracellular glucose may involve a number of processes. One consequence of excessive intracellular glucose levels is an increased rate of oxidative phosphorylation under hyperglycemic conditions, whereas another consequence is an increase in the metabolism of glucose to sorbitol by aldose reductase. In addition, hyperglycemia may result in the activation of NADPH oxidase, the production of superoxide anion, and hydrogen peroxide (H2O2). In this report, we investigate the mechanisms responsible for the H2O2 production that occurs as the consequence of hyperglycemia and the effect of H2O2 on the activity of the Na+/glucose cotransport system (SGLT) in primary cultures of renal proximal tubule cells (PTCs). When primary PTCs were cultured in the presence of high glucose, one consequence was that the Na+/glucose cotransport system was inhibited, as indicated by uptake studies utilizing alpha-methyl-D-glucoside (alpha-MG), a nonmetabolizable analog of D-glucose. Pretreatment of the cultures with either 1) aminoguanidine or pyridoxamine [inhibitors of the accumulation of advanced glycation end products (AGEs)], 2) rotenone (an inhibitor of the mitochondrial electron transport chain), or 3) apocynin or diphenylene iodonium (DPI; inhibitors of NADPH oxidase) blocked the observed changes that occurred as a consequence of the incubation of the PTCs with high glucose. Included among these changes were the observed increase in H2O2 levels, as well as an increase in lipid peroxide production, and a decrease both in the activity of catalase and in the level of glutathione (GSH), endogenous antioxidants. The high glucose-induced decrease in the level of the Na+/glucose cotransporter was similarly prevented by either aminoguanidine, rotenone, or apocynin. Thus the inhibitory effect of high glucose on both the level of the Na+/glucose cotransport system and the activity of the Na+/glucose cotransport system can be explained, at least in part, as being due to the effects of the H2O2, the consequent formation of AGEs, the increase in mitochondrial metabolism, and in NADPH oxidase activity in the PTCs. Other related changes observed in the PTCs that could be reversed by treatment with either aminoguanidine, pyridoxamine, rotenone, apocynin, or DPI included an increase in transforming growth factor-beta1 secretion and the activation of the NF-kappaB signal transduction pathway.

Electrical stimulation of transforming growth factor-beta 1 secretion by human dermal fibroblasts and the U937 human monocytic cell line.

The in vitro effects on human dermal fibroblasts and the U937 human monocytic cell line of three phases of electrical microcurrents generated by the ACE Stimulator were investigated. The growth and viability of growing and confluent dermal fibroblasts were not directly influenced by the separate microcurrent phases. One form of microcurrent (designated phase 1) stimulated both dermal fibroblasts and U937 cells to secrete transforming growth factor-beta 1 (TGF-beta 1), which is an important regulator of cell-mediated inflammation and tissue regeneration, but none of the three phases stimulated secretion of the pro-inflammatory cytokine interleukin-6 by U937 cells. The stimulation of TGF-beta 1 secretion in these experiments was not dramatic (a median increase over control levels of 20-30%), although it could be biologically significant.

Transforming Growth Factor-β1 Secretion From Murine Macrophages Is Released by In Vivo Ingestion of Apoptotic Cells

(CHEST 2001; 120:3S)

Epstein–Barr Virus Permissively Infects Human Syncytiotrophoblastsin Vitroand Induces Replication of Human T Cell Leukemia-Lymphoma Virus Type I in Dually Infected Cells

Epstein–Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1), as well as human T-cell leukemia-lymphoma virus type I (HTLV-I), may interact in the pathogenesis of human retroviral infections. The placental syncytiotrophoblast layer represents a barrier protecting the fetal compartment from exposure to retroviruses. We studied the interactions of EBV with HIV-1 and HTLV-I in human term syncytiotrophoblast cells to investigate the significance of double infections in transplacental transmission of human retroviruses. We found that syncytiotrophoblast cells could be productively infected with EBV. Dual infection of the cells with EBV and HTLV-I resulted in full replication cycle of otherwise latent HTLV-I. In contrast, the restricted permissiveness of syncytiotrophoblasts for HIV-1 was not influenced by coinfection of the cells with EBV. Infection of syncytiotrophoblast cells with EBV, but not HTLV-I, induced interleukin-2 and interleukin-6 secretion, and augmented secretion occurred on coinfection with both viruses. Coinfection of syncytiotrophoblast cells with EBV and HTLV-I induced tumor necrosis factor-β and transforming growth factor-β1 secretion, but infection with either virus alone did not lead to secretion of these cytokines. Permissive replication cycle of HTLV-I was induced by the EBV immediate-early gene product Zta. Pseudotype formation between EBV and HTLV-I in coinfected syncytiotrophoblast cells was not found. Our data suggest that activation of HTLV-I gene expression by EBV in coinfected syncytiotrophoblast cells may be a mechanism for transplacental transmission of HTLV-I.

Differential Transforming Growth Factor-β Secretion in Adenocarcinoma and Squamous Cell Carcinoma of the Uterine Cervix

Tumor cells from eight freshly isolated cervical cancers (i.e., four adenocarcinomas and four squamous carcinomas) were analyzed for their production of the immune-inhibitory cytokine transforming growth factor-β (TGF-β)in vitro.All fresh adenocarcinomas secreted significant levels of TGF-β (mean 397, range between 207 and 782 pg/ml/105cells/48 hr). In contrast, no detectable TGF-β was present in the supernatants from the four fresh squamous carcinoma cultures (P< 0.001). These data suggest that major differences in the secretion of the immunoinhibitory cytokine TGF-β exist between squamous cell carcinomas and adenocarcinomas of the uterine cervix. Furthermore, these findings suggest that at least some of the differences in the natural biologic behavior, as well as in the response to radiation treatment, between these two histologic types of cervical cancer could be related to differences in secretion of this immune-inhibitory cytokine.

Growth factors in the tumorigenicity of a brain tumor cell line

We studied a virally induced canine gliosarcoma brain tumor cell line capable of producing brain tumors in vivo following intracerebral inoculation of the cells into the brains of adult mongrel dogs. Cloned populations from this cell line differed in their ability to produce brain tumors in vivo. In vitro transforming growth factor-β secretion by each clone correlated with in vivo tumorigenicity. Mitogenic activity like that produced with platelet-derived growth factor was also secreted by each clone and correlated with in vivo tumorigenicity. Epidermal growth factor and transforming growth factor-α production was not detected in any of the clones of canine gliosarcoma cells. Although only some of the brain tumor cell clones were able to produce tumors in immunologically normal dogs, brain tumors were produced in all dogs pretreated with cyclosporin and in all “nude” mice, suggesting that tumor immunology plays an important role in this brain tumor model. In vitro cloning efficiency of each brain tumor cell clone also correlated with in vivo tumorigenicity. We postulate that transforming growth factor-β may both stimulate brain tumor cell growth and inhibit host antitumor immune surveillance.