Tumor Necrosis Factor-inducible Gene Research Articles

Tumor necrosis factor-inducible gene 6 protein ameliorates chronic liver damage by promoting autophagy formation in mice

Tumor necrosis factor-inducible gene 6 protein (TSG-6) has recently been shown to protect the liver from acute damage. However, the mechanism underlying the effect of TSG-6 on the liver remains unclear. Autophagy is a catabolic process that targets cell components to lysosomes for degradation, and its functions are reported to be dysregulated in liver diseases. Here we investigate whether TSG-6 promotes liver regeneration by inducing autophagic clearance in damaged livers. Mice fed a methionine choline-deficient diet supplemented with 0.1% ethionine (MCDE) for 2 weeks were injected with TSG-6 (the M+TSG-6 group) or saline (the M+V group) and fed with MCDE for 2 additional weeks. Histomorphological evidence of injury and increased levels of liver enzymes were evident in MCDE-treated mice, whereas these symptoms were ameliorated in the M+TSG-6 group. Livers from this group contained less active caspase-3 and more Ki67-positive hepatocytic cells than the M+V group. The autophagy markers ATG3, ATG7, LC3-II, LAMP2A and RAB7 were elevated in the M+TSG-6 group compared with those in the M+V group. Immunostaining for LC3 and RAB7 and electron microscopy analysis showed the accumulation of autophagy structures in the M+TSG-6 group. TSG-6 also blocked both tunicamycin- and palmitate-induced apoptosis of hepatocytes and increased their viability by inducing autophagy formation in these cells. An autophagy inhibitor suppressed TSG-6-mediated autophagy in the injured hepatocytes and livers of MCDE-treated mice. These results therefore demonstrate that TSG-6 protects hepatocytes from damage by enhancing autophagy influx and contributes to liver regeneration, suggesting that TSG-6 has therapeutic potential for the treatment of liver diseases.

Abstract 109: Tumor Necrosis Factor Inducible Gene 6 Protein (TSG-6) is Highly Expressed in Human Abdominal Aortic Aneurysms

Objective: The formation of an abdominal aortic aneurysm (AAA) is characterized by a dominance of pro-inflammatory forces that result in smooth muscle cell apoptosis, extra-cellular matrix degradation, and progressive diameter expansion. Additional defects in the anti-inflammatory response may also contribute to AAA progression, however have yet to be characterized robustly. Here, we describe the role of the anti-inflammatory cytokine TSG-6 (TNF-stimulated gene-6) in AAA formation. Methods: Blood and aortic tissue samples were collected from patients undergoing elective AAA screening and open surgical AAA repair. Aortic specimens collected were preserved for IHC or immediately assayed after tissue homogenization. Cytokine concentrations in tissue and plasma were assayed by ELISA. All immune cell populations were assayed using FACS analysis. In vitro, macrophage polarization from monocytes were performed with young, healthy donor PBMCs. Results: TSG-6 was found to be abnormally elevated in both the plasma and aorta of patients with AAA compared to healthy and risk-factor matched non-AAA donors. We observed the highest tissue concentration of TSG-6 in the less diseased proximal and distal shoulders compared to the central aspect of the aneurysm. IHC localized the majority of TSG-6 to the tunica media with minor expression in the tunica adventitia of the aortic wall. Higher concentrations of both M1 and M2 macrophages where also observed in the aortic wall, however M1/M2 ratios were unchanged from healthy controls. Additionally, we observed no difference in M1/M2 ratios in the peripheral blood of risk-factor matched non-AAA and AAA patients. Interesting, TSG-6 inhibited the polarization of the anti-inflammatory M2 phenotype in vitro . Conclusions: AAA formation results from an imbalance of inflammatory forces causing aortic wall infiltration of mononuclear cells leading to resultant vessel breakdown. From our results, we suggest TSG-6 is elevated in the AAA patient as a compensatory anti-inflammatory feedback mechanism. However, it’s effects may be abrogated by defects in CD44, its cognate receptor or downstream signaling pathways, future areas for investigation.

TSG-6- a double-edged sword for osteoarthritis

TSG-6- a double-edged sword for osteoarthritis

Dermal Fibroblasts Promote Alternative Macrophage Activation Improving Impaired Wound Healing

Tight control of inflammation is required for tissue repair and wound healing and depends on alternative polarization of macrophages as checkpoint for inflammatory resolution. Its perturbations lead to impaired regeneration. Administration of cells/cell factors capable of reversing inflammation and rescuing alternative polarization could be promising for treating inflammatory diseases. We show that human dermal fibroblasts (dFb) are ideal candidates for such a task by demonstrating a new function of these cells, which is modulating macrophage polarization. Coculture of dFb with human monocytes invitro or injection of dFb into mice with thioglycollate-induced peritonitis favors alternative macrophage activation and reduces inflammation by releasing tumor necrosis factor-inducible gene 6 protein and Cox-2 products. Silencing these factors in dFb abolishes the reported effects, demonstrating their importance for immunomodulation. Importantly, in a model of delayed wound healing due to prolonged inflammation (db/db mice), administration of dFb improves defective tissue repair with augmentation of alternative macrophage polarization and inflammation resolution. Human dFb are low immunogenic cells, easy to obtain, and can be expanded extensively invitro conserving their immunomodulatory capacity; this, together with our findings, suggests that dFb might represent an alternative for cell-based therapies of conditions characterized by excessive inflammation and delayed tissue repair.

Effects of Anomalin, Hyuganin C and Pteryxin on Phorbol Ester- or Tumor Necrosis Factor-induced Gene Expression, Production and Secretion of MUC5AC Mucin from airway Epithelial Cells

We investigated whether anomalin, hyuganin C or pteryxin affects airway MUC5AC mucin gene expression, production and secretion induced by phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-α (TNF-α) from NCIH292 cells. Cells were pretreated with each agent for 30 min and then stimulated with PMA or TNF-α for 24h. The three compounds inhibited the gene expression, production and secretion of MUC5AC mucin from NCI-H292 cells. This result suggests that anomalin, hyuganin C and pteryxin can inhibit the secretion, production and gene expression of mucin induced by PMA or TNF-α, through directly acting on airway epithelial cells.

A Multicenter Study of Early Anti-inflammatory Treatment in Patients With Acute Anterior Cruciate Ligament Tear

Background: It is increasingly recognized that biochemical abnormalities of the joint precede radiographic abnormalities of posttraumatic osteoarthritis (PTOA) by as much as decades. A growing body of evidence strongly suggests that the progression from anterior cruciate ligament (ACL) injury to PTOA is multifactorial, involving the interplay between biomechanical disturbances and biochemical homeostasis of articular cartilage. Purpose: The purposes of this randomized study using an acute ACL injury model were to (1) evaluate the natural progression of inflammatory and chondrodegenerative biomarkers, (2) evaluate the relationship between subjective reports of pain and inflammatory and chondrodegenerative biomarkers, and (3) determine if postinjury arthrocentesis and corticosteroid injection offer the ability to alter this biochemical cascade. Study Design: Randomized controlled trial; Level of evidence, 2. Methods: A total of 49 patients were randomized to 4 groups: group 1 (corticosteroid at 4 days after ACL injury, placebo injection of saline at 2 weeks), group 2 (placebo at 4 days after ACL injury, corticosteroid at 2 weeks), group 3 (corticosteroid at both time intervals), or a placebo group (saline injections at both time intervals). Patient-reported outcome measures and synovial biomarkers were collected at approximately 4 days, 11 days, and 5 weeks after injury. The change between the time points was assessed for all variables using Wilcoxon tests, and the relationship between changes in outcome scores and biomarkers were assessed by calculating Spearman ρ. Outcomes and biomarkers were also compared between the 4 groups using Kruskal-Wallis tests. Results: No adverse events or infections were observed in any study patients. With the exception of matrix metalloproteinase 1 (MMP-1) and tumor necrosis factor–inducible gene 6 (TSG-6), chondrodegenerative markers worsened over the first 5 weeks while all patient-reported outcomes improved during this time, regardless of treatment group. Patient-reported outcomes did not differ between patients receiving corticosteroid injections and the placebo group. However, increases in C-telopeptide of type II collagen (CTX-II), associated with collagen type II breakdown, were significantly greater in the placebo group (1.32 ± 1.10 ng/mL) than in either of the groups that received the corticosteroid injection within the first several days after injury (group 1: 0.23 ± 0.27 ng/mL [P = .01]; group 3: 0.19 ± 0.34 ng/mL [P = .01]). Conclusion: PTOA begins at the time of injury and results early on in dramatic matrix changes in the knee. However, it is encouraging that early intervention with an anti-inflammatory agent was able to affect biomarkers of chondral degeneration. Should early intervention lead to meaningful changes in either the onset or severity of symptomatic PTOA, the current treatment paradigm for patients with ACL injury may have to be restructured to include early aspiration and intra-articular intervention. Trial Registration: ClinicalTrials.gov identifier: NCT01692756.

Tumor necrosis factor-inducible gene 6 protein: A novel neuroprotective factor against inflammation-induced developmental brain injury

Inflammation is an important factor contributing to developmental brain injury in preterm infants. Although tumor necrosis factor-inducible gene 6 protein (TSG-6) has immunomodulatory effects in several inflammatory conditions of adult animals, nothing is currently known about the role of TSG-6 in the developing brain, its impact on perinatal inflammation and its therapeutic potential. The aim of the current work was 1) to characterize the developmental expression of TSG-6 in the newborn rat brain, 2) to evaluate the impact of LPS exposure on TSG-6 expression and 3) to assess the therapeutic potential of exogenous TSG-6 administration. Brain hemispheres of healthy Wistar rats (postnatal day 1–postnatal day 15 (P1–P15)) were evaluated with regard to the physiological expression of TSG-6. LPS-treated rats (0.25mg/kg LPS i.p. on P3) were analyzed for inflammation-induced changes in TSG-6 and cytokine expression. To evaluate whether exogenous recombinant human (rh)TSG-6 affects inflammation-induced brain injury, newborn Wistar rats, exposed to LPS on P3, were treated with rhTSG-6 i.p. (four repetitive doses of 2.25mg/kg every 12h, first dose 3h before LPS injection). PCR, Western blotting and multiplex ELISA were performed according to standard protocols. TSG-6 is physiologically expressed in the developing brain with a linear increase in expression from P1 to P15 at the mRNA level. At P6, regional differences in TSG-6 expression in the cortex, thalamus and striatum were detected at mRNA and protein level. Furthermore, TSG-6 gene expression was significantly increased by inflammation (induced by LPS treatment). Combined treatment with LPS and TSG-6 vs. LPS exposure alone, resulted in significant down-regulation of cleaved caspase-3, a marker of apoptosis and neuronal plasticity. In addition, several inflammatory serum markers were decreased after TSG-6 treatment. Finally, TSG-6 is physiologically expressed in the developing brain. Changes of TSG-6 expression associated with inflammation suggest a role of TSG-6 in neuroinflammation. Reduction of cleaved caspase-3 by TSG-6 treatment demonstrates the putative neuroprotective potential of exogenous TSG-6 administration in inflammation-induced developmental brain injury.

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor.

Hyaluronan Rafts on Airway Epithelial Cells

Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyte-adhesive hyaluronan (HA) cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables but occur in distinct sheets or rafts that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-α-inhibitor (IαI) proteoglycan, which has two heavy chains (HC1 and HC2) on its chondroitin sulfate chain. The transesterification transfer of HCs from chondroitin sulfate to HA is mediated by tumor necrosis factor-induced gene 6 (TSG-6), which is up-regulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of IαI, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-IαI) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage of naïve mice and were observed on their apical ciliated surfaces. Thus, these leukocyte-adhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions.

Tumor necrosis factor-inducible gene 6 promotes liver regeneration in mice with acute liver injury.

IntroductionTumor necrosis factor-inducible gene 6 protein (TSG-6), one of the cytokines released by human mesenchymal stem/stromal cells (hMSC), has an anti-inflammatory effect and alleviates several pathological conditions; however, the hepatoprotective potential of TSG-6 remains unclear. We investigated whether TSG-6 promoted liver regeneration in acute liver failure.MethodsThe immortalized hMSC (B10) constitutively over-expressing TSG-6 or empty plasmid (NC: Negative Control) were established, and either TSG-6 or NC-conditioned medium (CM) was intraperitoneally injected into mice with acute liver damage caused by CCl4. Mice were sacrificed at 3 days post-CM treatment.ResultsHigher expression and the immunosuppressive activity of TSG-6 were observed in CM from TSG-6-hMSC. The obvious histomorphological liver injury and increased level of liver enzymes were shown in CCl4-treated mice with or without NC-CM, whereas those observations were markedly ameliorated in TSG-6-CM-treated mice with CCl4. Ki67-positive hepatocytic cells were accumulated in the liver of the CCl4 + TSG-6 group. RNA analysis showed the decrease in both of inflammation markers, tnfα, il-1β, cxcl1 and cxcl2, and fibrotic markers, tgf-β1, α-sma and collagen α1, in the CCl4 + TSG-6 group, compared to the CCl4 or the CCl4 + NC group. Protein analysis confirmed the lower expression of TGF-β1 and α-SMA in the CCl4 + TSG-6 than the CCl4 or the CCl4 + NC group. Immunostaining for α-SMA also revealed the accumulation of the activated hepatic stellate cells in the livers of mice in the CCl4 and CCl4 + NC groups, but not in the livers of mice from the CCl4 + TSG-6 group. The cultured LX2 cells, human hepatic stellate cell line, in TSG-6-CM showed the reduced expression of fibrotic markers, tgf-β1, vimentin and collagen α1, whereas the addition of the TSG-6 antibody neutralized the inhibitory effect of TSG-6 on the activation of LX2 cells. In addition, cytoplasmic lipid drops, the marker of inactivated hepatic stellate cell, were detected in TSG-6-CM-cultured LX2 cells, only. The suppressed TSG-6 activity by TSG-6 antibody attenuated the restoration process in livers of TSG-6-CM-treated mice with CCl4.ConclusionsThese results demonstrated that TSG-6 contributed to the liver regeneration by suppressing the activation of hepatic stellate cells in CCl4-treated mice, suggesting the therapeutic potential of TSG-6 for acute liver failure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0019-z) contains supplementary material, which is available to authorized users.

Adipose tissue-derived stem cells suppress acute cellular rejection by TSG-6 and CD44 interaction in rat kidney transplantation.

In addition to its abundance and easy accessibility, adipose tissue yields more potent immunoregulatory stem cells (adipose tissue-derived stem cells, ADSCs) than does bone marrow. However, the beneficial effects of ADSCs on alloreactivity are scarcely known. This study evaluated the beneficial effects of ADSCs in rat kidney transplantation and analyzed the underlying molecular mechanism. Dark Agouti rat kidneys were transplanted into Lewis rats. Autologous ADSCs (2×10) were injected through the left renal artery of the donors before the nephrectomy (ADSCs group). Graft survival, histologic changes, and the expression of several cytokines and proteins were assessed. In an in vitro experiment, the immunosuppressive capacity of ADSCs was tested in a mixed lymphocyte reaction. Histologic findings of the ADSCs group revealed a reduced rejection grade, whereas the number of infiltrated CD4/CD8 T cells was also significantly decreased as compared to the control. Relative to these findings, injection of ADSCs led to a significantly prolonged mean graft survival compared with the control. In vitro, autologous ADSCs dose-dependently suppressed alloreactive lymphocytes. Moreover, ADSCs increased the level of tumor necrosis factor-inducible gene 6 protein (TSG-6) in mixed lymphocyte reaction, which has an anti-inflammatory capacity. Recombinant TSG-6 markedly suppressed alloreactive T cells through downregulating CD44, which may lead to the suppression of T-cell activation and infiltration into allografts. Our findings clearly showed that ADSCs attenuated acute rejection by secreting TSG-6 as well as through direct cell interaction. These findings contribute to the clinical application of these cells in solid organ transplantation.

Evaluating Potential Therapies in a Mouse Model of Focal Retinal Degeneration with Age-related Macular Degeneration (AMD)-Like Lesions

Although the mouse has no macula leutea, its neuroretina and retinal pigment epithelium (RPE) can develop lesions mimicking certain features of age-related macular degeneration (AMD). Differences between the Ccl2 and Cx3cr1 double deficient mouse on Crb1rd8(rd8) background (DKOrd8) and the Crb1rd8 mouse in photoreceptor and RPE pathology, as well as ocularA2E contents and immune responses, show that DKOrd8 recapitulates some human AMD-like features in addition to rd8 retinal dystrophy/degeneration. Different therapeutic interventions have been demonstrated to be effective on the AMD-like features of DKOrd8 mice. The use of the DKOrd8 model and C57BL/6N (wild type, WT) mice as group controls (4 groups) to test treatments such as high omega-3 polyunsaturated fatty acid (n-3) diet has, for example, shown the beneficial effect of n-3 on AMD-like lesions by anti-inflammatory action of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The use of self-control in the DKOrd8 mouse by treating one eye and using the contralateral eye as the control for the same mouse allows for appropriate interventional experiments and evaluates various novel therapeutic agents. Three examples will be briefly presented and discussed: (1) tumor necrosis factor-inducible gene 6 recombinant protein (TSG-6) arrests the AMD-like lesions via modulation of ocular immunological gene expression, e.g., Il-17a; (2) adeno-associated virus encoding sIL-17R (AAV2.sIL17R) stabilizes the AMD-like lesions; and (3) pigment epithelium-derived factor (PEDF) ameliorates the AMD-lesions by its anti-inflammatory, anti-apoptotic and neuroprotective roles. Therefore, the DKOrd8 mouse model can be useful and appropriate for therapeutic compound screening in the management of human AMD.

309 Tumor Necrosis Factor-Inducible Gene 6 Protein: A Novel Neuroprotective Factor Against Inflammatory Developmental Brain Injury

Background and AimsAn important factor of developmental brain injury is inflammation. It has been shown that tumor necrosis factor-inducible gene 6 protein (TSG-6) has anti-inflammatory effects in several inflammatory conditions....

Anti-inflammatory recombinant TSG-6 stabilizes the progression of focal retinal degeneration in a murine model

BackgroundInflammatory responses are detected in the retina of patients with age-related macular degeneration and Ccl2-/-/Cx3cr1-/- mice on rd8 background,(Ccl2-/-/Cx3cr1-/- mice) a model that develops progressive age-related macular degeneration-like retinal lesions including focal photoreceptor degeneration, abnormal retinal pigment epithelium and A2E accumulation. Tumor necrosis factor-inducible gene 6 protein is an anti-inflammatory protein and has been shown to improve myocardial infarction outcome and chemically injured cornea in mice by suppressing inflammation. In this study, we evaluated the effect of an intravitreous injection of recombinant TSG-6 on the retinal lesions of Ccl2-/-/Cx3cr1-/- mice.MethodsRecombinant TSG-6 (400 ng) was administered by intravitreous injection into the right eye of six-week-old Ccl2-/-/Cx3cr1-/- mice. Their left eye was injected with phosphate-buffered saline as a control. Funduscopic pictures were taken before injection and sequentially once a month after injection. The mice were killed two months after injection and the ocular histology examined. Retinal A2E, a major component of lipofuscin, was measured by high performance liquid chromatography. The microarray of ocular mRNA of 92 immunological genes was performed. The genes showing differentiated expression in microarray were further compared between the injected right eye and the contralateral (control) eye by [real-time quantitative reverse transcription polymerase chain reaction] qRT-PCR.ResultsThe continuous monitoring of the fundus for two months showed a slower progression or alleviation of retinal lesions in the treated right eyes as compared with the untreated left eyes. Among 23 pairs of eyes, the lesion levels improved in 78.3%, stayed the same in 8.7% and progressed in 13.0%. Histology confirmed the clinical observation. Even though there was no difference in the level of A2E between the treated and the untreated eyes, microarray analysis of 92 immune genes showed that IL-17a was substantially decreased after the treatment. Expression of TNF-α showed a similar pattern to IL-17a. The results were consistent in duplicated arrays and confirmed by qRT-PCR.ConclusionsWe concluded that intravitreous administration of recombinant TSG-6 might stabilize retinal lesions in Ccl2-/-/Cx3cr1-/- mice on rd8 background. Modulation of ocular immunological gene expressions, especially IL-17a, could be one of the mechanisms.

Targeted Cardiac Overexpression of A20 Improves Left Ventricular Performance and Reduces Compensatory Hypertrophy After Myocardial Infarction

A20 was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells. As an inhibitor of nuclear factor-kappaB signaling, A20 protects against apoptosis, inflammation, and cardiac hypertrophy. In the present study, we tested the hypothesis that cardiac-specific overexpression of A20 could protect the heart from myocardial infarction. We investigated the role of constitutive human A20 expression in acute myocardial infarction using a transgenic model. Transgenic mice containing the human A20 gene under the control of the alpha-myosin heavy chain promoter were constructed. Myocardial infarction was produced by coronary ligation in A20 transgenic mice and control animals. The extent of infarction was then quantified by 2-dimensional and M-mode echocardiography and by molecular and pathological analyses of heart samples in infarct and remote heart regions 7 days after myocardial infarction. Constitutive overexpression of A20 in the murine heart resulted in attenuated infarct size and improved cardiac function 7 days after myocardial infarction. Significantly, we found a decrease in nuclear factor-kappaB signaling and apoptosis, as well as proinflammatory response, cardiac remodeling, and interstitial fibrosis, in noninfarct regions in the hearts of constitutive A20-expressing animals compared with control animals. Cardiac-specific overexpression of A20 improves cardiac function and inhibits cardiac remodeling, apoptosis, inflammation, and fibrosis after acute myocardial infarction.

306 GENERATION OF HUMAN A20 GENE-TRANSGENIC PORCINE FETAL FIBROBLASTS FOR SOMATIC CELL NUCLEAR TRANSFER

The aim of this project was to produce transgenic pigs with improved features in xenotransplantation, by expressing the human A20 gene to modulate the acute vascular rejection (AVR) reaction ocurring after porcine-to-human xenotransplantation. The A20 gene was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells (Opipari AW et al. 1990 J. Biol. Chem. 25, 14 705–14 708). The gene is both anti-apoptotic and anti-inflammatory in endothelial cells (Ferran C 2006 Transplantation 82(1 Suppl.), S36–S40) and could thus prevent endothelial cell activation leading to AVR. The hA20-expression vector driven by the CAGGS hybrid promoter (chicken β-actin–rabbit β-globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFF) derived from German Landrace porcine fetal explant cultures. Transfection of 3 � 106 cells was accomplished at 450 V and 350 µF with 10 µg of plasmid DNA. Then, G418-resistant cell clones (800 µg mL–1) were screened by PCR with hA20-specific primers for hA20 integration. Eighty clones were A20-positive in PCR screening from 4 rounds of transfection. One cell clone was verified by DNA sequencing and subsequently used as donor cells in somatic cell nuclear transfer. One hundred sixty-nine embryos were transferred to 2 synchronized peripuberal German Landrace gilts, respectively. Ultrasound examination of recipient sows on Day 22 after embryo transfer confirmed established pregnancies in both recipients. One pregnancy was allowed to go to term and 7 healthy piglets were born, whereas the second pregnancy was terminated on Day 70 of pregnancy for detailed expression analysis of the 8 isolated fetuses. Results showed that the A20 vector can be integrated in PFF, and A20-transgenic PFF can be successfully used in somatic cell nuclear transfer to establish pregnancies. Further analysis will focus on the expression levels and patterns in A20-positive cell clones and the biological function in transgenic piglets. Functional assays will be conducted in vitro and in vivo. We thank Prof. Beyaert of Ghent University, Belgium for providing us with the expression vector pCAGGSEhA20.

Anti-Inflammatory and Chondroprotective Effect of TSG-6 (Tumor Necrosis Factor-α-Stimulated Gene-6) in Murine Models of Experimental Arthritis

Tumor necrosis factor-alpha (TNF-alpha)-stimulated gene-6 (TSG-6) is up-regulated by various cytokines and growth factors. TSG-6 binds to hyaluronan in inflamed synovial tissue and forms a complex with a serine protease inter-alpha-trypsin inhibitor (IalphaI), increasing the protease inhibitory effect of IalphaI >100-fold. The TSG-6/IalphaI complex then blocks serine proteases, including the plasminogen-plasmin activation, probably the most important component in the activation processes of matrix metalloproteinases. To gain insight into the mechanisms of TSG-6 action in arthritis, we have used an autoimmune murine model (proteoglycan-induced arthritis) for systemic, and a monoarticular form of arthritis (antigen-induced arthritis) for local treatment of arthritis with recombinant mouse TSG-6 (rmTSG-6). Intravenous injection of rmTSG-6 induced a dramatic reduction of edema in acutely inflamed joints by immobilizing CD44-bound hyaluronan and, in long-term treatment, protected cartilage from degradation and blocked subchondral and periosteal bone erosion in inflamed joints. The intra-articular injection of a single dose (100 microg) of rmTSG-6 exhibited a strong chondroprotective effect for up to 5 to 7 days, preventing cartilage proteoglycan from metalloproteinase-induced degradation. In contrast, rmTSG-6 did not postpone the onset, nor reduce the incidence of arthritis. We were unable to detect any significant differences between control and rmTSG-6-treated animals when various serum markers (including pro- and anti-inflammatory cytokines, auto- and heteroantibody productions) or antigen-specific T-cell responses were compared, nor when the expressions of numerous cell surface receptors or adhesion molecules were measured. TSG-6 seems to play a critical negative regulatory feed-back function in inflammation, especially in arthritic processes.

Glucocorticoids repress NF-kappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell.

Glucocorticoids (GCs) are used to combat inflammatory diseases. Their beneficial effect relies mainly on the inhibition of NF-kappaB- and/or AP-1-driven proinflammatory gene expression. Previously, we have shown that GCs repress tumor necrosis factor-induced IL-6 gene expression by an NF-kappaB-dependent nuclear mechanism without changing the DNA-binding capacity of NF-kappaB or the expression levels of the cytoplasmic inhibitor of NF-kappaB (IkappaB-alpha). In the present work, we investigate the effect of GC repression on different natural and/or recombinant NF-kappaB-driven reporter gene constructs in the presence of increasing amounts of various coactivator molecules, such as CREB-binding protein (CBP), p300, and SRC-1. We found that GCs maintain their repressive capacities, irrespective of the amount of cofactor present in the cell. Similar results were obtained for the reciprocal transrepression of a GC receptor (GR) element-driven reporter gene by p65. We demonstrate that neither the expression levels of p65 and CBP nor their physical association are affected by activated GR. Using Gal4 chimeras, we show that repression by GCs is specific for p65-mediated transactivation, ruling out competition for limiting nuclear factors as the major underlying mechanism of gene repression. In addition, the transactivation potential of a point-mutated Gal4-p65 variant with a decreased CBP interaction capability is still repressed by GR. Finally, we present evidence that the specificity of GC repression on p65-driven gene expression is codetermined by the TATA box context.

Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on transcription factor NF-kappa B.

In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-alpha (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 mumol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-kappa B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-kappa B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-kappa B-independent mechanism(s).

Four different classes of inhibitors of receptor-mediated endocytosis decrease tumor necrosis factor-induced gene expression in human endothelial cells.

We have investigated the relationship between receptor-mediated endocytosis of TNF and TNF-induced gene expression in cultured human endothelial cells. Exposure of cells to hypertonicity, to cytoplasmic acidification, to treatment with phenylarsine oxide, or to treatment with primary amines such as putrescine or dansylcadaverine each inhibited receptor-mediated endocytosis by 30 to 75%, as measured by uptake of acetylated Dil-low density lipoprotein or of 125I-TNF. All four treatments also inhibited TNF-induced surface expression of ELAM-1 by 50 to 100%. Among these four treatments, only hypertonicity inhibited pinocytosis, as measured by uptake of fluorescein-BSA, and only phenylarsine oxide irreversibly inhibited protein synthesis, as measured by [35S]methionine incorporation. Notably, acidification or treatment with primary amines selectively inhibited the response to TNF, compared with the response to PMA, a drug that induces ELAM-1 through a pathway that bypasses surface receptors. Primary amines, which can be used for sustained periods under physiologic culture conditions without causing toxicity, were investigated further. Pretreatment of endothelial cells with 10 mM putrescine or 100 microM dansylcadaverine also inhibited TNF induction of ICAM-1 expression and VCAM-1 expression. Primary amines also inhibited IL-1-induced increases in ELAM-1, ICAM-1, and VCAM-1 measured 4 to 6 h after treatment and inhibited IFN-beta- and IFN-gamma-mediated induction of class I MHC molecules and IFN-gamma-mediated induction of class II MHC molecules measured 72 h after treatment with cytokine. Levels of mRNA encoding cytokine-inducible molecules were also selectively reduced by primary amines. A constitutively expressed surface molecule, gp96, was not affected in the same cells. These data are consistent with a role for receptor-mediated endocytosis in TNF-mediated gene induction and suggest a new potential target for anti-inflammatory therapy.