Generation and Regulation of CD8＋ Regulatory T Cells

Research into the suppressive activity of CD4＋FoxP3＋ T regulatory cells （Treg） has defined a sublineage of CD4＋ cells that contribute to self-tolerance and resistance to autoimmune disease. Much less attention has been given to the potential contribution of regulatory sublineages of CD8＋ cells. Analysis of a small fraction of CD8＋ cells that target autoreactive CD4＋ cells through recognition of the MHC class Ib molecule Qa-1 in mouse and HLA-E in human has revitalized interest in CD8＋ Treg. Here we summarize recent progress and future directions of research into the role of this CD8＋ sublineage in resistance to autoimmune disease. Cellular ＆ Molecular Immunology. 2008; 5（6）：401-406.     

Rapamycin Inhibits Transforming Growth Factor β1-Induced Fibrogenesis in Primary Human Lung Fibroblasts

Purpose

The present study was designed to determine whether rapamycin could inhibit transforming growth factor β1 (TGF-β1)-induced fibrogenesis in primary lung fibroblasts, and whether the effect of inhibition would occur through the mammalian target of rapamycin (mTOR) and its downstream p70S6K pathway.

Materials and Methods

Primary normal human lung fibroblasts were obtained from histological normal lung tissue of 3 patients with primary spontaneous pneumothorax. Growth arrested, synchronized fibroblasts were treated with TGF-β1 (10 ng/mL) and different concentrations of rapamycin (0.01, 0.1, 1, 10 ng/mL) for 24 h. We assessed m-TOR, p-mTOR, S6K1, p-S6K1 by Western blot analysis, detected type III collagen and fibronectin secreting by ELISA assay, and determined type III collagen and fibronectin mRNA levels by real-time PCR assay.

Results

Rapamycin significantly reduced TGF-β1-induced type III collagen and fibronectin levels, as well as type III collagen and fibronectin mRNA levels. Furthermore, we also found that TGF-β1-induced mTOR and p70S6K phosphorylation were significantly down-regulated by rapamycin. The mTOR/p70S6K pathway was activated through the TGF-β1-mediated fibrogenic response in primary human lung fibroblasts.

Conclusion

These results indicate that rapamycin effectively suppresses TGF-β1-induced type III collagen and fibronectin levels in primary human lung fibroblasts partly through the mTOR/p70S6K pathway. Rapamycin has a potential value in the treatment of pulmonary fibrosis.     

uPAR Induces Expression of Transforming Growth Factor β and Interleukin-4 in Cancer Cells to Promote Tumor-Permissive Conditioning of Macrophages

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor β and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell–like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow–derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor β was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.It is well established that certain chronic infections and inflammation predispose to the development of malignancy.^1–3 Once cancer develops, inflammatory cells that infiltrate the tumor may promote disease progression.^4–6 This process is mediated by bidirectional paracrine pathways involving cancer and inflammatory cells. Growth factors and cytokines released by cancer cells are immunosuppressive, and also condition inflammatory cells so that these cells release mediators that support cancer cell growth, survival, metastasis, and angiogenesis.^7–10 Inflammatory cell conditioning is prevalent in breast cancer. These tumors include large numbers of macrophages, dendritic cells, mast cells, and T cells, and the extent to which the tumor is infiltrated by these inflammatory cells correlates with the incidence of metastasis.^11–13 A high density of tumor-associated macrophages (TAMs) is also correlated with higher breast cancer tumor grade and decreased relapse-free and overall survival.^14–17Although macrophages express a wide spectrum of phenotypic properties, these cells are frequently categorized as classically activated (M1) or alternatively activated (M2).^18–21 In response to pathogens, tissue damage, and Th1 cytokines such as IFN-γ and TNF-α, M1-polarized macrophages release cytotoxic compounds and proteins, including nitric oxide, reactive oxygen species, and proinflammatory cytokines (including IL-12, IL-23, and TNF-α). M2-polarized macrophage have been classified into a number of subcategories; in many contexts, these cells demonstrate enhanced activity in the resolution of inflammation, tissue remodeling, and healing.^18–21 Arginase 1 (Arg1), which is expressed selectively by M2-polarized macrophages, diverts substrate from the enzyme systems that produce cytotoxic levels of nitric oxide.^22,23 In general, it is thought that TAMs, which have been conditioned by cancer cells to express tumor-permissive gene products, demonstrate characteristics in common with M2-polarized macrophages, although a recent report highlights phenotypic differences.^18,19,24 Cell-signaling systems in tumor cells that promote the ability of these cells to regulate macrophage phenotype remain incompletely understood.In many forms of cancer, expression of the urokinase receptor [urokinase plasminogen activator receptor (uPAR)] correlates with poor prognosis and shortened survival.^25–28 Originally, the activity of uPAR in cancer was attributed to its ability to bind the serine protease, urokinase-type plasminogen activator (uPA), and activate a cascade of extracellular proteases involved in matrix remodeling and cell migration through tissue boundaries. The current understanding, however, is that uPAR also is a cell-signaling receptor that activates diverse signaling pathways.²⁹ Although uPAR may signal autonomously when expressed at high levels, uPA binding to uPAR robustly activates cell signaling even when the cell-surface abundance of uPAR is low.^29–32 uPAR-initiated cell signaling promotes cancer cell survival, release from states of dormancy, migration, epithelial–mesenchymal transition, cancer stem cell–like properties, and metastasis independently of protease activation.^33–38Here, we show that in multiple forms of cancer, including breast cancer, pancreatic cancer, and glioblastoma (GBM), uPAR expression promotes the ability of the cancer cells to M2-polarize co-cultured macrophages. The mediators that are released selectively by uPAR-expressing cancer cells to regulate macrophage phenotype may vary across different cancer cells; however, we provide evidence that both TGF-β and IL-4 are involved. The ability of cancer-cell uPAR to promote conditioning of inflammatory cells in the tumor microenvironment is a novel mechanism by which uPAR may promote cancer progression.     

Hydrogen-Rich Saline Ameliorates Allergic Rhinitis by Reversing the Imbalance of Th1/Th2 and Up-Regulation of CD4+CD25+Foxp3+Regulatory T Cells,Interleukin-10, and Membrane-Bound Transforming Growth Factor-β in Guinea Pigs

It is well known that CD4+CD25+Foxp3+Treg cells play an important role in the development of allergic rhinitis (AR); the defect of cell numbers and functions contribute to AR. Hydrogen has been proven effective in alleviating symptoms of AR. We herein aim to verify the protective effects of hydrogen on CD4+CD25+Foxp3+Treg cells in guinea pigs with AR and to explore the effect of hydrogen-rich saline (HRS) on CD4+CD25+Foxp3+Treg cells in animals with AR and investigate the underlying anti-inflammatory mechanism. Eighteen guinea pigs were randomly divided into three groups (control group/AR group/AR-HRS group). The guinea pigs were injected with hydrogen-rich saline (AR-HRS group) for 10 days after sensitization. The control group was injected with an equal volume of normal saline. The number of sneezes, degree of runny nose, and nasal-rubbing movements were scored. Peripheral blood eosinophil count was recorded. The proportions of Th1/Th2 of the peripheral blood and the CD4+CD25+Foxp3+T cells in the CD4+T cells of the spleen and peripheral blood were determined by flow cytometry. The content of interleukin (IL)-10 and transforming growth factor (TGF)-β in the serum was detected by enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expression of Foxp3, IL-10, and TGF-β were determined by Western blot, immunofluorescence, and real-time PCR analysis, respectively. Scores of symptoms, number of eosinophils,and nasal mucosa damage were dramatically reduced after HRS treatment. HRS increased the expression of Foxp3, IL-10, TGF-β, and number of CD4+CD25+Foxp3+Treg cells, which were reduced in AR. HRS also revised the dysregulation of Th1/Th2 balance. Both the number and biological activity of CD4+CD25+Foxp3+Treg cells increased with up-regulation of Th1/Th2 after HRS administration. HRS could play a protective role in attenuating AR through improving the proportion and functions of CD4+CD25+Foxp3+Treg cells.     

Involvement of Transforming Growth Factor β and its Type 1 Receptor in the Development of Breast Cancer

Introduction of recent achievements of molecular biology into clinical practice contributes significantly to both prevention and early detection of breast cancer in women and development of new approaches, including genotyping, to screening for breast cancer. Screening for genetic polymorphisms in genes TGFβ1 and TGFβR1 reveals carriers of sporadic breast cancer. This provides an opportunity to reassess the role and place of preventive measures, also including surgical method, in the fight against breast cancer.     

Effects of Transforming Growth Factor-β1 on Proliferation of Smooth Muscle Cells in Human Aortic Intima and Human Promonocytic Leukemia THP-1 Cells

We studied the effects of transforming growth factor on proliferation of cultured smooth muscle cells from human aortic intima and proliferation and differentiation of human leukemia THP-1 promonocytes. Transforming growth factor inhibited proliferation of these cells, but stimulated differentiation of THP-1 cells. Therefore, transforming growth factor probably modulates proliferation and differentiation of smooth muscle cells and monocytes/macrophages involved in the pathogenesis of atherosclerotic damages.     

Neutralization of Transforming Growth Factor β1 Augments Hepatitis C Virus-Specific Cytotoxic T Lymphocyte Induction in Vitro

In hepatitis C virus (HCV) infection, TGF-1 is upregulated in the liver and may be involved in the pathogenesis of chronic liver disease. TGF-1 is also produced by activated T cells and acts as a potent immunosuppressor. The aim of this study was to investigate the roles of TGF-1 in HCV-specific cytotoxic T lymphocyte (CTL) induction and enhance their killer activity by TGF-1 modulation. We generated anti-HCV CTL from peripheral blood mononuclear cells from HLA-A2 patients under stimulation with the HCV-core peptide having the HLA-A2.1 binding motif. The lytic activities of CTL or precursor frequency (CTLpf) generated with or without anti-TGF-p antibody were compared. To optimize the IL-2 dose for CTL induction, low (50 U/ml) and high (500 U/ml) doses were tested and the lytic activities were compared. TGF-1 amounts in the supernatants were assessed by enzyme-linked immunosorbent assay and by their growth inhibitory effect on mink lung epithelial cells. CTL activity was enhanced by anti-TGF- antibody in a dose-dependent manner but CTLpf did not significantly change. A high dose of IL-2 reduced the activity to 45% of that observed with a low dose, whereas TGF-1 increased as the dose of IL-2 increased. Exogenous IL-10 reversed the inhibitory effect of a high dose of IL-2 on the killing activity by reducing TGF-1 mRNA expression in T cells and its production. These results demonstrated that endogenous TGF-1 is an autocrine suppressor in CTL induction in vitro. Therefore, the blockade of endogenous TGF-1 could enhance the killing potential of anti-HCV CTL.     

Increased Levels of Transforming Growth Factor β1 and Basic Fibroblast Growth Factor in Patients on CAPD: A Study During Non-Infected Steady State and Peritonitis

Long-term influence of continuous ambulatory peritoneal dialysis (CAPD) on concentrations of transforming growth factor 1 (TGF-1) and basic fibroblast growth factor (bFGF) in the peritoneal effluent, and the effect of peritonitis on these cytokines were investigated. TGF-1 and bFGF were assayed in effluent samples from dialysate bags collected during the initial week of treatment with CAPD and at 5 months. To determine the effect of peritonitis, dialysate bags were collected on admission to the hospital and on days 3 and 10 and also during non-infected steady state. Serum was drawn prior to infection and on days 1 and 10. TGF-1 increased more than threefold during the longitudinal follow-up period, median concentrations of 35 pg/ml to 106 pg/ml (P < 0.05). No change in bFGF was seen during this initial 5 months. TGF-1 was increased on the first day of peritonitis (median concentration 169 pg/ml) and reached its maximum on day 3 of infection, (median concentration 216 pg/ml) (P < 0.05 vs non-infected state, median concentration 39 pg/ml). Basic FGF reached a maximum on day three of infection (median concentration 7.7 pg/ml; P = 0.01 vs non-infected state) and then slowly declined. In conclusion, TGF-1 is influenced by CAPD treatment per se, and together with bFGF is increased during peritonitis, indicating its importance in the peritoneum and its potential involvement in the development of tissue fibrosis and eventually ultrafiltration failure.     

Type 1 CD8^＋ T Cells are Superior to Type 2 CD8^＋ T Cells in Tumor Immunotherapy due to Their Efficient Cytotoxicity, Prolonged Survival and Type 1 Immune Modulation

CD8^＋ cytotoxic T （Tc） cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8^＋ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8^＋ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin （OVA）-pulsed dendritic cell （DCovA）-activated CD8^＋ type 1 Tc （Tcl） cells secreting IFN-T, and CD8^＋ type 2 Tc （Tc2） cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor （TCR） transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tcl and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8^＋ Tc2, Tcl cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8^＋ Tcl and Tc2 cells skewed the phenotype of CD4^＋ T cells toward Thl and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8^＋ Tcl and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8^＋ T cells for adoptive T cell therapy of cancer.     

Effects of Transforming Growth Factor-βl and Phorbol Ester on PALI-1 and PA Genes in Human Lung Cells

Abstract

Transforming growth factor-β (TGF-β) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-β and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-β in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-β involves PKC. TGF-β1 and PMA increased expression of TGF-βl mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-β2 and TGF-β3 were more complex; while TGF-β2 and TGF-p3β mRNAs increased transiently in response to TGF-p1 in NHBE cells and TGF-β3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCLH727 where TGF-pZ mRNA increased dramatically. TGF-β1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contmst, while TGF-βl also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-β1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-βl, PAI-1 and PA by TGF-β1 and PMA in NSCLC, but differential regulation of the genes for PAL1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-β1, PAI-1 and PA to TGF-βl and PMA were inhibited by the serind threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-β1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-p responses by inhibiting a protein serindthmnine kinase(s). Because the effects of TGF-p and PMA on the different TGF-p isoforms, PA, PA1 and fibronectin in NHBE and NSCLC cells are complex, our data suggest that there are distinct mechanisms for controlling the different TGF-p isoforms, PA, PA1 and extracellular matrix proteins in normal lung and lung cancer cells.     

Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases.     

Transforming Growth Factor β-1 Stimulates Profibrotic Epithelial Signaling to Activate Pericyte-Myofibroblast Transition in Obstructive Kidney Fibrosis

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti–TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis.Pericytes are mesenchyme-derived perivascular cells attached to the abluminal surface of capillaries.¹ They share developmental origins with fibroblasts, and there may be plasticity between pericytes attached to capillaries and fibroblasts embedded in adjacent collagenous matrix; however, unlike fibroblasts, pericytes have vital functions in regulating microvascular stability, angiogenesis, capillary permeability, capillary flow, and capillary basement membrane synthesis.¹ We have previously shown that pericytes are the major sources of scar-producing myofibroblasts during kidney injury, and we have identified adult kidney pericytes and perivascular fibroblasts are derived from Foxd1-expressing progenitors, positive for collagen I(α1)-GFP (Coll-GFP⁺), platelet-derived growth factor receptor β (PDGFR-β⁺), and CD73 (CD73⁺) and negative for α smooth muscle actin (α-SMA⁻) and CD45 (CD45⁻).^2–4 Recently, spinal cord pericytes were identified as major progenitors of scar tissue in the central nervous system, intestinal pericytes as a source of myofibroblasts in models of colitis, and hepatic stellate cells, the major precursor of myofibroblasts in liver disease, have been determined to be specialized pericytes of the hepatic sinusoid,^5–8 indicating that pericytes may represent myofibroblast precursors in many organs. Many independent studies support the notion of perivascular resident mesenchymal cells, not injured tubular epithelial cells, as the major source of myofibroblasts in kidneys.^9–12Prompted by the newly identified role for these perivascular cells in the pathogenesis of kidney fibrosis, we earlier investigated the cellular crosstalk that regulates pericyte detachment from capillaries and regulates the transition of pericytes to myofibroblasts.^13–15 Our investigations so far have focused on pericyte-endothelial crosstalk, because pericytes form direct communications with endothelial cells of peritubular capillaries at peg and socket junctions, where direct cell-cell signaling has been thought to occur.^13–20 We have recently shown that Coll-GFP⁺ kidney pericytes function identically to brain pericytes in migrating to and stabilizing capillary networks, functions that require expression of tissue inhibitor of metalloproteinase 3 (TIMP-3).¹⁵ These pericyte functions are lost when Coll-GFP⁺ pericytes transition to myofibroblasts.¹⁵ Furthermore, we reported that endothelial activation at vascular endothelial cell growth factor (VEGF) receptor 2 and PDGFR-β signaling by pericytes are two critical signaling pathways that link endothelial activation with pericyte transition to myofibroblasts.¹⁴ Our studies showed that these signaling events alone are sufficient to drive microvascular rarefaction, inflammation, and fibrosis in models of kidney disease.¹⁴ These findings are striking, because during embryonic and fetal microvascular development these same signaling pathways are critical in normal formation of the vasculature, indicating that dysregulation of signaling pathways between endothelium and pericytes is central to kidney pathogenesis.Nonetheless, studies unequivocally show that the injured tubular epithelium can directly trigger interstitial fibrosis. For example, overexpression of VEGF-A in adult kidney epithelium is sufficient to drive fibrosis, and cell cycle arrest of the kidney proximal epithelium at the G2/M checkpoint is also sufficient to drive fibrosis.^21,22 Therefore, epithelial signaling events must somehow be transmitted across the tubular basement membrane to pericytes to drive interstitial fibrosis. These obscure molecular signaling events are the focus of the studies we report here.In previous investigations of embryonic microvascular development, endothelial cells have been shown to be a source of both PDGF and transforming growth factor β-1 (TGF-β1), cytokines that regulate pericyte attachment, differentiation, and angiogenesis.^17,23,24 Moreover, genetic inactivation of either TGFB1 or of genes encoding its receptors in mice leads to vascular defects and embryonic lethality.^17–19 TGF-β1 is thus a cytokine with a profound effect on microvascular development and angiogenesis.In adult kidney injury, although endothelial cells produce PDGF and TGF-β1 in fibrosing kidneys, injured epithelial cells are a major source of these cytokines, and the TGF-β1 activator integrin αvβ6 is restricted to kidney epithelium.^13,25–29 Increased TGF-β1 expression by epithelium is accompanied by activation of intracellular signaling pathways and downstream effectors in the epithelium itself.^30,31 Blocking TGF-β1 and its downstream effectors can attenuate kidney injury and fibrosis,^30–33 whereas transgenic overexpression of TGF-β1 in kidney epithelial cells is sufficient to trigger interstitial kidney fibrosis in the absence of migration of epithelial-derived cells into the interstitium.^34,35 Therefore, epithelial transgenic overexpression of TGF-β1, which stimulates epithelial cell dedifferentiation and autophagy, must stimulate pericyte to myofibroblast transition by epithelial cell to pericyte crosstalk.³⁴ Our aim in the present study was to identify the mechanism by which TGF-β1 signaling from injured tubular epithelial cells can activate pericytes to drive progressive kidney fibrosis.     

Differential Sensitivity of Naïve and Memory Subsets of Human CD8+ T Cells to TNF-α-Induced Apoptosis

In this investigation, we have examined the relative sensitivity of human naïve, central memory (T_CM), and two types of effector memory CD8+ T cells (T_EM and T_EMRA) to TNF-α-induced apoptosis. Our data show that naïve and T_CM CD8+ T cells were sensitive, whereas T_EM and T_EMRA CD8+ T cells were relatively resistant to TNF-a-induced apoptosis. The apoptosis profile correlated with the activation of caspase-8 and caspase-3. However, no correlation was observed between relative sensitivity of four CD8 + T cell subsets to apoptosis and the expression of TNFR-I or TNFR-II. T_EM and T_EMRA CD8+ T cells displayed increased phosphorylation of IKKα/β and IκB and increased NF-κB activity as compared to naïve and T_CM CD8+ T cells. Bcl-2, Bcl-x_L and FLIP_L expression was higher and Bax expression was lower in T_EM and T_EMRA CD8+ T cells as compared to naïve and T_CM CD8+ T cells. These data suggest that signaling molecules downstream of TNFRs may be responsible for differential sensitivity among subsets of CD8+ T cells to TNF-α-induced apoptosis.     

Mannose-6-Phosphate/IGF-II Receptors Mediate the Effects of IGF-1-Induced Latent Transforming Growth Factor β1 on Expression of Type I Collagen and Collagenase in Dermal Fibroblasts

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor β1 (LTGF-β1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-β1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-β1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-β1, collagenase and the pro α1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-β1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro α1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-β1 neutralizing antibody at a concentration of 0.5–2.0 μg/ml. As the TGF-β1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 ± 38 vs 81.8 ± 13, p < 0.05) in mRNA for the pro α1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-β1 and the pro α1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-β1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-β1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-β1 and that the matrix-modulating autocrine effects of LTGF-β1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.