Cell intrinsic TGF-beta 1 regulation of B cells

TGF-beta family cytokines play multiple roles in immune responses. TGF-beta1-null mice suffer from multi-organ infiltration that leads to their premature death. T cells play a central role in the TGF-beta1 phenotype, as deficiency of TGF-beta1 only in T cells reproduces the lethal phenotype. Although it is known that TGF-beta1 controls B cells isotype switch and homeostasis, the source responsible for this control has not been characterized. Because of the major role that T cells play in regulating B cell responses, we addressed the T cell dependency of the TGF-beta1 control of B cells. The analysis of T cell-deficient, TGF-beta1 knockout mice and the production of chimeras in which B but not T cells lacked TGF-beta1 allowed us to show that B cells are controlled in part by cell autonomous production of TGF-beta1.     

TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells

TGF-beta 1 is critical for differentiation of epithelial-associated dendritic Langerhans cells (LC). In accordance with the characteristics of in vivo LC, we show that LC obtained from human monocytes in vitro in the presence of TGF-beta 1 1) express almost exclusively intracellular class II Ags, low CD80, and no CD83 and CD86 Ags and 2) down-regulate TNF-RI (p55) and do not produce IL-10 after stimulation, in contrast to dermal dendritic cells and monocyte-derived dendritic cells. Surprisingly, while LC exhibit E-cadherin down-regulation upon exposure to TNF-alpha and IL-1, TGF-beta 1 prevents the final LC maturation in response to TNF-alpha, IL-1, and LPS with respect to Class II CD80, CD86, and CD83 Ag expression, loss of FITC-dextran uptake, production of IL-12, and Ag presentation. In sharp contrast, CD40 ligand cognate signal induces full maturation of LC and is not inhibited by TGF-beta 1. The presence of emigrated immature LCs in human reactive skin-draining lymph nodes provides in vivo evidence that LC migration and final maturation may be differentially regulated. Therefore, due to the effects of TGF-beta 1, inflammatory stimuli may not be sufficient to induce full maturation of LC, thus avoiding potentially harmful immune responses. We conclude that TGF-beta 1 appears to be responsible for both the acquisition of LC phenotype, cytokine production pattern, and prevention of noncognate maturation.     

TGF-β1 regulation of dendritic cells

Dendritic cells (DCs) represent antigen-presenting cell (APC) populations in lymphoid and nonlymphoid organs which are considered to play key roles in the initiation of antigen-specific T-cell proliferation. According to current knowledge, the net outcome of T-cell immune responses seems to be significantly influenced by the activation stage of antigen-presenting DCs. Several studies have shown that transforming growth factor-beta 1 (TGF-β1) inhibits in vitro activation and maturation of DCs. TGF-β1 inhibits upregulation of critical T-cell costimulatory molecules on the surface of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-β1 on effector T lymphocytes, inhibitory effects of TGF-β1 at the level of APCs may critically contribute to previously characterized immunosuppressive effects of TGF-β1. In contrast to these negative regulatory effects of TGF-β1 on function and maturation of lymphoid tissue type DCs, certain subpopulations of immature DCs in nonlymphoid tissues are positively regulated by TGF-β1 signaling. In particular, epithelial-associated DC populations seem to critically require TGF-β1 stimulation for development and function. Recent studies established that TGF-β1 stimulation is absolutely required for the development of epithelial Langerhans cells (LCs) in vitro and in vivo. Furthermore, TGF-β1 seems to enhance antigen processing and costimulatory functions of epithelial LCs.     

TGF-beta 1 reciprocally controls chemotaxis of human peripheral blood monocyte-derived dendritic cells via chemokine receptors

We examined the effect of TGF-beta 1 on the chemotactic migratory ability of human monocyte-derived dendritic cells (DCs). Treatment of immature DCs with TGF-beta 1 resulted in increased expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXC chemokine receptor-4 (CXCR-4), which were concomitant with enhanced chemotactic migratory responses to their ligands, RANTES (for CCR-1, CCR-3, and CCR-5), macrophage-inflammatory protein-3 alpha (MIP-3 alpha) (for CCR-6), or stromal cell-derived growth factor-1 alpha (for CXCR-4). Ligation by TNF-alpha resulted in down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3 alpha, and stromal cell-derived growth factor-1 alpha, whereas this stimulation up-regulated the expression of CCR-7 and the chemotactic ability for MIP-3beta. Stimulation of mature DCs with TGF-beta 1 also enhanced TNF-alpha-induced down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and chemotaxis to their respective ligands, while this stimulation suppressed TNF-alpha-induced expression of CCR-7 and chemotactic migratory ability to MIP-3 beta. Our findings suggest that TGF-beta 1 reversibly regulates chemotaxis of DCs via regulation of chemokine receptor expression.     

Functional study of immature dendritic cells co-transfected with IL-10 and TGF-beta 1 genes in vitro

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previous studies suggest that immature dendritic cells (imDCs) might be involved in the induction of peripheral T cell tolerance. While interleukin-10 (IL-10) functions at different levels of the immune response, transforming growth factor-beta 1 (TGF-beta 1) is considered to be a key factor in immune tolerance. In this study, we investigated the effects of immature DC (imDC) co-transfected with IL-10 and TGF-beta 1 genes (IL-10-TGF-beta 1-imDC) on inducing immune tolerance. Moreover, we compared the effects of IL-10-TGF-beta 1-imDC with IL-10 transfected imDC (IL-10-imDC) and TGF-beta 1-transfected imDC (TGF-beta 1-imDC), respectively. IL-10-TGF-beta 1-imDC resulted in the down-regulation of MHC class II, CD80 and CD86. IL-10-TGF-beta 1-imDC could induce T cell hyporesponsiveness, and was reluctant to proliferate. IL-10-TGF-beta 1-imDC was more effective than IL-10-imDC and TGF-beta 1-imDC, respectively. In summary, co-expression of IL-10 and TGF-beta 1 affected the immunity of imDCs and enhanced their tolerogenicity. It might be a promising therapy for donor-specific tolerance after organ transplantation.     

Splenic stromal microenvironment negatively regulates virus-activated plasmacytoid dendritic cells through TGF-beta

Plasmacytoid dendritic cells (pDCs) secrete large amounts of IFN-alpha upon exposure to virus, subsequently promoting and regulating innate and adaptive immune responses. However, little is known about the functional regulation of virus-activated pDCs after they exert functions in secondary lymph organs. Our previous studies show that splenic stromal microenvironment can down-regulate the T cell response by inducing generation of regulatory myeloid dendritic cells; therefore, we wondered whether the splenic stromal microenvironment can regulate the function of virus-activated pDCs. In this study, we provide evidences that the splenic stromal microenvironment can chemoattract vesicular stomatitis virus (VSV)-activated pDCs via stromal cell-derived factor 1 (SDF-1), inhibit the secretion of IFN-alpha, IL-12, TNF-alpha, and expression of I-Ab, CD86, CD80, and CD40 by VSV-activated pDCs, and subsequently inhibit VSV-infected pDCs to activate NK cell IFN-gamma production and cytotoxicity. Stroma-derived TGF-beta participates in the negative regulation of VSV-activated pDCs. Therefore, we demonstrate that splenic stromal microenvironment negatively regulates the virus-activated pDCs through TGF-beta, outlining an additional mechanistic explanation for maintenance of immune homeostasis.     

Restoration of TGF-beta regulation of plasminogen activator inhibitor-1 in Smad3-restituted human choriocarcinoma cells

Proliferation, migration, and invasiveness of the normal placental extravillous trophoblast (EVT) cells are negatively regulated by transforming growth factor-beta (TGF-beta), whereas malignant EVT (JAR and JEG-3 choriocarcinoma) cells are resistant to TGF-beta. These malignant cells were found to have lost the expression of Smad3. Present study examined whether Smad3 restitution in JAR cells could restore TGF-beta response. We produced a stable Smad3 cDNA-transfected clone (JAR-smad3/c) which exhibited further upregulation of Smad3 in the presence of TGF-beta1. Since anti-invasive effects of TGF-beta in the normal EVT cells were shown to be mediated in part by plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA), we compared the expression of PAI-1 and uPA in the normal EVT, JAR, and JAR-smad3/c cells in the presence or absence of TGF-beta1. The basal levels of PAI-1 mRNA and secreted PAI-1 and uPA proteins were found to be very low in JAR and JAR-smad3/c cells, as compared to the normal EVT cells. However, TGF-beta1 upregulated PAI-1 and downregulated uPA in JAR-smad3/c cells, but not in JAR cells. Thus, resistance of choriocarcinoma cells to anti-invasive effects of TGF-beta may, at least in part, be due to loss of Smad3 expression.     

Functional regulation of monocyte-derived dendritic cells by microRNAs

Dendritic cells (DCs) as a rare type of leukocytes play an important role in bridging the innate and adaptive immune system. A subset of DCs, monocyte-derived dendritic cells (moDCs), exists in very low numbers at steady state but become abundant in inflammatory states. These inflammation-associated DCs are potent producers of pro-inflammatory cytokines and potent inducers of T helper differentiation. They behave as a “double-edge” sword so that they not only mediate protective immunity but also immuno-pathology. It is still incompletely understood how their function is regulated. Emerging evidence indicates that microRNAs (miRNAs), as a new class of gene regulators, potently regulate the function of moDCs. Here we summarize recent progress in this area.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.     

IFN regulation and functions in myeloid dendritic cells

A central issue in dendritic cells (DC) biology is to understand how type I IFNs modulate the immuno-regulatory properties of DC. In this review I will address this issue in light of the recent experimental evidence on the expression and function of these cytokines in myeloid DC. This knowledge may have important therapeutic implications in infectious and neoplastic diseases and open new perspectives in the use of IFNs as vaccine adjuvants and in the development of DC-based vaccines.     

Functional involvement of E-cadherin in TGF-beta 1-induced cell cluster formation of in vitro developing human Langerhans-type dendritic cells

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-beta 1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-beta 1 costimulation (basic cytokine combination GM-CSF plus TNF-alpha, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-beta 1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a-, lysozyme+, CD14+). TGF-beta 1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-beta 1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-beta 1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.     

Genomics of TGF-beta1 signaling in stem cell commitment and dendritic cell development

Dendritic cells are professional antigen presenting cells and central for establishing and maintaining immunity and immunological tolerance. They develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Dendritic cell development and function are regulated by specific cytokines, including transforming growth factor type beta1 (TGF-beta1). Our previous work demonstrated the importance of TGF-beta1 signaling for dendritic cell development and subset specification. Here, we used genome-wide gene expression profiling with DNA microarrays to investigate the activity of TGF-beta1 on gene expression in dendritic cell development. This study identified specific gene categories induced by TGF-beta1 with an impact on dendritic cell biology.     

Signalling and regulation of collagen I synthesis by ET-1 and TGF-beta1

Endothelin-1 (ET-1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET-1 signals are transduced by two receptor subtypes, the ETA- and ETB-receptors which activate different Galpha proteins. Here, we investigated the expression of both ET-receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA-receptor is the major ET-receptor subtype expressed. To determine further signalling intermediates, we inhibited Galphai and three phospholipases. Pharmacologic inhibition of Galphai, phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD), but not of phospholipase Cbeta, abolished the increase in collagen I by ET-1. Inhibition of all phospholipases revealed similar effects on TGF-beta1 induced collagen I synthesis, demonstrating involvement of PC-PLC and PLD in the signalling pathways elicited by ET-1 and TGF-beta1. ET-1 and TGF-beta1 each stimulated collagen I production and in an additive manner. ET-1 further induced connective tissue growth factor (CTGF), as did TGF-beta1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF-beta1 treatment, ET-1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET-1 treatment. Coincidentally at 5 days of permanent ET-1 stimulation, a switch in ET-receptor subtype expression to the ETB-receptor was observed. We conclude that the signalling pathways induced by ET-1 and TGF-beta1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long-time stimulation by ET-1 resulted in a changed ET-receptor subtype ratio and in a biphasic CTGF induction.     

NF-AT-mediated expression of TGF-beta1 in tolerant T cells

During T cell development in the thymus, a certain population of self-reactive thymocytes differentiates into regulatory T cells that suppress otherwise harmful self-reactive T cells. In transgenic mice expressing both TCR that specifically recognizes moth cytochrome c and the moth cytochrome c ligand, a large proportion of CD4+ T cells expresses CD25 and secretes TGF-beta1 upon Ag stimulation. Because TGF-beta1 expression by these T cells can be decreased by cyclosporin A, a NF-AT inhibitor, NF-AT-mediated TGF-beta1 expression in T cells was addressed by characterizing a NF-AT response element in the TGF-beta1 promoter. Analysis of the mouse TGF-beta1 promoter (-1799 to +793) in transfection experiments in T cell 68-41 hybridoma cells detected NF-AT binding sites at positions +268 and +288 in the proximal promoter region. Binding of NF-AT to this region was detected only in tolerant CD4+ T cells, but not in fully activated CD4+ T cells by chromatin immunoprecipitation assays. Activation of these NF-AT sites was sufficient to induce TGF-beta1 promoter activity; however, additional signaling due to full Ag stimulation blocked NF-AT-mediated TGF-beta1 expression. This suppression of the TGF-beta1 promoter is mediated by the -1079 to -406 region, in which deletion of a GATA-binding motif at position -821 abrogates NF-AT-mediated activation of the TGF-beta1 promoter. Therefore, TGF-beta1 expression in T cells is controlled by multiple regulatory factors that have distinct functions in response to partial or full TCR activation.