Tumor Necrosis Factor-stimulated Gene-6 Research Articles

Constitutive Expression of Pentraxin 3 (PTX3) Protein by Human Amniotic Membrane Cells Leads to Formation of the Heavy Chain (HC)-Hyaluronan (HA)-PTX3 Complex

Heavy chain (HC)-hyaluronan (HA), a complex formed by the covalent linkage between HC1 from the inter-α-trypsin inhibitor (IαI) and HA, purified from the human amniotic membrane (AM), is responsible for the anti-inflammatory, antiscarring, and antiangiogenic actions of the AM. This HC-HA complex is produced by constitutive expression of TNF-stimulated gene 6 and endogenous production of IαI by AM cells. Pentraxin 3 (PTX3), a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens, also helps stabilize HC-HA to ensure female fertility. Here we noted strong positive PTX3 staining in the AM epithelium and compact stroma. PTX3 was constitutively expressed and secreted by cultured AM epithelial and stromal cells and, further, greatly up-regulated by TNF and IL-1β. Using an agarose overlay to trap the HA-containing matrix, the HC-HA-PTX3 complex was formed, as analyzed by Western blot analysis, by AM cells but not human skin fibroblasts, despite being cultured in the presence of serum and TNF. However, exogenous PTX3 helps human skin fibroblasts form the HC-HA-PTX3 complex with an agarose overlay. Furthermore, PTX3 can be coimmunoprecipitated with the HC-HA complex from agarose-overlaid AM cell extracts by an anti-human IαI antibody. Such a HC-HA-PTX3 complex can be reconstituted in vitro and exhibit similar effects as those reported for AM HC-HA-PTX3 on polarization of M2 macrophages. The tight binding between PTX3 and AM HC-HA withstands four runs of CsCl ultracentrifugation in the presence of 4 m GnHCl. These results indicate that PTX3 is constitutively expressed and secreted by AM cells as an integral component of the AM HC-HA-PTX3 complex and contributes to the biological function of AM HC-HA-PTX3.

Modification of Hyaluronan by Heavy Chains of Inter-α-Inhibitor in Idiopathic Pulmonary Arterial Hypertension

We previously reported an altered hyaluronan (HA) metabolism in idiopathic pulmonary arterial hypertension (IPAH) lung tissue and cultured smooth muscle cells. Hyaluronan was present in the smooth muscle cell layer surrounding the pulmonary vasculature and in plexigenic lesions. Additionally, cultured pulmonary artery smooth muscle cells produced spontaneous HA "cable" structures, without additional stimuli, that were leukocyte-adhesive. We now present evidence that the HA that accumulates in IPAH plexigenic lesions is a pathological form of HA in which heavy chains (HCs) from the serum-derived proteoglycan inter-α-inhibitor are covalently attached to the HA backbone to form a pathological HC-HA complex. CD45-positive leukocytes were identified within these HC-HA matrices. Elevated mRNA levels of the enzyme that transfers HCs to HA, known as tumor necrosis factor-stimulated gene 6, were detected in IPAH lung tissue.

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan: USE OF DEFINED OLIGOSACCHARIDES TO PROBE STRUCTURE AND FUNCTION

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was (13)C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.

Mesenchymal stromal (stem) cells suppress pro-inflammatory cytokine production but fail to improve survival in experimental staphylococcal toxic shock syndrome

BackgroundToxic shock syndrome (TSS) is caused by an overwhelming host-mediated response to bacterial superantigens produced mainly by Staphylococcus aureus and Streptococcus pyogenes. TSS is characterized by aberrant activation of T cells and excessive release of pro-inflammatory cytokines ultimately resulting in capillary leak, septic shock, multiple organ dysfunction and high mortality rates. No therapeutic or vaccine has been approved by the U.S. Food and Drug Administration for TSS, and novel therapeutic strategies to improve clinical outcome are needed. Mesenchymal stromal (stem) cells (MSCs) are stromal cells capable of self-renewal and differentiation. Moreover, MSCs have immunomodulatory properties, including profound effects on activities of T cells and macrophages in specific contexts. Based on the critical role of host-derived immune mediators in TSS, we hypothesized that MSCs could modulate the host-derived proinflammatory response triggered by Staphylococcal enterotoxin B (SEB) and improve survival in experimental TSS.MethodsEffects of MSCs on proinflammatory cytokines in peripheral blood were measured in wild-type C57BL/6 mice injected with 50 μg of SEB. Effects of MSCs on survival were monitored in fatal experimental TSS induced by consecutive doses of D-galactosamine (10 mg) and SEB (10 μg) in HLA-DR4 transgenic mice.ResultsDespite significantly decreasing serum levels of IL-2, IL-6 and TNF induced by SEB in wild-type mice, human MSCs failed to improve survival in experimental TSS in HLA-DR4 transgenic mice. Similarly, a previously described downstream mediator of human MSCs, TNF-stimulated gene 6 (TSG-6), did not significantly improve survival in experimental TSS. Furthermore, murine MSCs, whether unstimulated or pre-treated with IFNγ, failed to improve survival in experimental TSS.ConclusionsOur results suggest that the immunomodulatory effects of MSCs are insufficient to rescue mice from experimental TSS, and that mediators other than IL-2, IL-6 and TNF are likely to play critical mechanistic roles in the pathogenesis of experimental TSS.

Modulation of tumor necrosis factor-stimulated gene-6 (TSG-6) expression in human endometrium

The human endometrium undergoes repetitive, cyclical changes under the influence of endocrine signals (estrogen and progesterone). In particular, the extensive tissue remodeling, angiogenesis and leukocyte infiltration that occur during decidualization of the endometrium give rise to an environment that is permissive to blastocyst attachment. However, it is now well established that crosstalk (via paracrine signals) between the trophoblast and the endometrium is also a key for successful implantation. Microarray studies have identified TSG-6 as a gene with elevated expression in endometrial stromal cells following the exposure to trophoblast and immune cell products. TSG-6 is an inflammation-associated protein which acts as a potent inhibitor of neutrophil extravasation and also plays important roles in matrix remodeling, e.g., by promoting hyaluronan (HA) cross-linking and down-regulating the protease network. Female TSG-6 (-/-) mice are infertile and this has been attributed to their inability to ovulate; however, the importance of TSG-6 in implantation has not been directly investigated. Real-time PCR, as well as immunofluorescence staining was performed on endometrial biopsies of proliferative and secretory phase samples. In addition stromal cell cultures of human endometrium were used to assess the influence of different stimulating factors on the TSG-6 gene and protein expression via real-time PCR and ELISA. Herein demonstrate that TSG-6 mRNA is expressed by the endometrium during the proliferative and secretory phases of the menstrual cycle. We also show that conditioned media from placental tissues induce rapid upregulation of TSG-6 mRNA expression and sustained protein secretion, with evidence that TNF is an important factor in this effect. Furthermore, we demonstrate changes in protein expression in the mid-secretory phase in women affected by recurrent abortions. These data suggest that TSG-6 expression might be essential in endometrial matrix organization and feto-maternal communication during the implantation process.

Inter-α-inhibitor Impairs TSG-6-induced Hyaluronan Cross-linking

Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

Human Mesenchymal Stem Cells Overexpressing the IL-33 Antagonist Soluble IL-1 Receptor–Like–1 Attenuate Endotoxin-Induced Acute Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by pulmonary edema attributable to alveolar epithelial-interstitial-endothelial injury, associated with profound inflammation and respiratory dysfunction. The IL-33/IL-1 receptor-like-1 (ST2) axis plays a key role in the development of immune-inflammatory responses in the lung. Cell-based therapy has been recently proposed as an effective alternative for the treatment of ALI and ARDS. Here, we engineered human adipose tissue-derived mesenchymal stem cells (hASCs) overexpressing soluble IL-1 receptor-like-1 (sST2), a decoy receptor for IL-33, in order to enhance their immunoregulatory and anti-inflammatory properties when applied in a murine ALI model. We administered both hASCs and hASC-sST2 systemically at 6 hours after intranasal LPS instillation, when pathological changes had already occurred. Bioluminescence imaging, immunohistochemistry, and focused transcriptional profiling confirmed the increased presence of hASCs in the injured lungs and the activation of an immunoregulatory program (CXCR-4, tumor necrosis factor-stimulated gene 6 protein, and indoleamine 2,3-dioxygenase up-regulation) in these cells, 48 hours after endotoxin challenge. A comparative evaluation of hASCs and the actions of hASC-sST2 revealed that local sST2 overproduction by hASC-sST2 further prevented IL-33, Toll-like receptor-4, IL-1β, and IFN-γ induction, but increased IL-10 expression in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in protein content, differential neutrophil counts, and proinflammatory cytokine (TNF-α, IL-6, and macrophage inflammatory protein 2) concentrations in bronchoalveolar lavage fluid. In addition, hASC-sST2-treated ALI lungs showed preserved alveolar architecture, an absence of apoptosis, and minimal inflammatory cell infiltration. These results suggest that hASCs genetically engineered to produce sST2 could become a promising therapeutic strategy for ALI/ARDS management.

FRI0351 Modulation of the innate humoral immunity in patients with refractory takayasu arteritis treated with biologic agents.

BackgroundTakayasu arteritis (TA) is a relapsing inflammatory large-vessel panarteritis resulting in vessel stenosis, occlusion or aneurysm formation1,2. Medical therapy with steroids and immunosuppressive agents is the cornerstone of TA treatment...

Abstract 3915: Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc.

Abstract A new investigational therapeutic agent, pegylated recombinant human hyaluronidase PH20 (PEGPH20) is a currently under clinical development for the treatment of tumors that accumulate hyaluronan (HA), a nonsulfated glycosaminoglycan and a major constituent of the extracellular matrix (ECM) of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival in multiple malignancies. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. To facilitate non-invasive clinical selection of patients with HA-rich malignancies, and confirm PEGPH20 enzymatic targeting of these malignancies, we have developed a soluble recombinant HA binding protein by fusing a tumor necrosis factor-stimulated gene-6 protein (TSG6) fragment, mutated at the heparin-binding site, with human IgG1 Fc (TSG6dHep-Fc). In preclinical proof-of-concept studies, recombinant TSG6dHep-Fc was labeled with the near-infrared fluorophore DyLight755 (TSG6dHep-FcDL755) and administered IV to the following xenograft peritibial athymic mouse models: HA-rich pancreatic BxPC3, HA-poor prostate Du145 and HA-rich prostate Du145/HAS2. TSG6dHep-FcDL755 HA binding was imaged via fluorescence (IVIS Lumina II, Caliper Life Sciences, Inc.). Systemic/dermal HA binding was weakly present, but easily removed via background subtraction to allow tumor intensity quantification. In the HA-rich xenograft models, pancreatic BxPC3 and prostate Du145/HAS2, TSG6dHep-FcDL755 strongly labeled the peritibial tumors, peaking at 2 days post administration and was completely absent at day 10 post administration. In contrast, the HA-poor prostate Du145 tumors were very weakly labeled, making background subtraction of systemic/dermal HA binding difficult. In separate experiments, mice were treated with a single dose of PEGPH20 (4.5 mg/kg, IV) or vehicle prior to TSG6dHep-FcDL755 imaging. In all groups dosed with PEGPH20, TSG6dHep-FcDL755 signal intensity was extremely low and appeared to be limited to non-specific, whole body labeling. These results suggest that imaging with labeled TSG6dHep-Fc could be a useful tool for selecting patients with HA-rich malignancies, thereby enabling clinicians to noninvasively identify patients who might benefit from therapies that target HA and the ECM. Citation Format: Xiaoming Li, Lei Huang, Ge Wei, Ryan J. Osgood, Qiping Zhao, Ping Jiang, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3915. doi:10.1158/1538-7445.AM2013-3915

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation *

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition, Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6(-/-) mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6(-/-) mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6(-/-) mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.

257 EFFECTS OF FIBROBLAST GROWTH FACTOR 8 ON CUMULUS EXPANSION AND NUCLEAR MATURATION IN BOVINE CUMULUS - OOCYTE COMPLEXES

Recent data indicate that fibroblast growth factor (FGF) signalling regulates oocyte developmental competence. Fibroblast growth factor 10 enhanced nuclear maturation, cumulus expansion, and embryo development in cattle (Zhang et al. 2010 Reproduction 140, 815–826). Like FGF10, FGF8 is expressed in the bovine oocyte, but whereas FGF10 activates FGF receptors (FGFR) 1B and 2B with higher affinity, FGF8 preferentially activates FGF receptor (FGFR) 2C, FGFR3C, and FGFR4. The involvement of FGF8 in the regulation of bovine cumulus–oocyte complex (COC) maturation has remained unknown. This study aimed to assess the effects of FGF8 supplementation in the in vitro maturation medium on nuclear maturation, degree of cumulus expansion, and expression of the genes necessary for expansion in bovine COC. Groups of 20 immature COC (grades 1 and 2) aspirated from 3- to 8-mm follicles were cultured in 200-µL drops of TCM-199 supplemented with FSH (1 µg mL–1), LH (10 IU mL–1), pyruvate (22 µg mL–1), amikacin (75 µg mL–1), and graded doses of recombinant human FGF8 (Peprotech, Rocky Hill, NJ, USA; 0, 1, 10, and 100 ng mL–1) for 22 h at 38.5°C and 5% CO2. After culture, COC were visually classified according to the degree of cumulus expansion (grades 1 to 3, indicating absent, moderate, and full expansion, respectively). Oocytes were mechanically separated from cumulus cells and stained with Hoechst 33342 to assess meiosis progression. Total RNA was extracted from cumulus cells using RNeasy (Qiagen, Venlo, the Netherlands), and 100 ng of RNA was reverse-transcribed using Omniscript (Qiagen). Expression levels of messenger RNA encoding genes necessary for cumulus expansion [prostaglandin endoperoxide synthase 2 (PTGS2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene-6 protein (TSG6)] were assessed by real-time PCR, with cyclophilin (CYCA) as the housekeeping gene. Data were derived from 5 replicates. Maturation and expansion data were transformed to arcsine, and gene expression data were log transformed. Effects of treatments were tested by ANOVA, and means were compared with the Tukey-Kramer honestly significant difference test. The FGF8 at 10 and 100 ng mL–1 reduced the proportion of oocytes reaching metaphase II (70, 64.8, 52.8, and 36% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.005) and increased the proportion of oocytes in metaphase I at 22 h of culture (30, 35.2, 47.2, and 64% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.004). Fibroblast growth factor 8 did not affect the degree of cumulus expansion as visually assessed. However, FGF8 at 10 and 100 ng mL–1 increased the messenger RNA abundance of PTGS2 [P = 0.0002; relative values (±SEM) of 0.69 ± 0.10, 0.63 ± 0.11, 1.56 ± 0.44, and 1.67 ± 0.20 for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively] and HAS2 [P = 0.0002; relative values (±SEM) of 1.38 ± 0.15, 1.37 ± 0.24, 3.58 ± 0.61, and 4.14 ± 0.27 for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively] in cumulus cells. In conclusion, the present data suggest the involvement of FGF8 in the mechanisms regulating transcription of expansion-inducing genes in cattle. In contrast with previous findings with FGF10, FGF8 inhibited nuclear maturation, suggesting different actions for different FGF in the regulation of COC maturation. Further research is needed to clarify the roles of FGF8 in the bovine COC. Supported by FAPESP.

Heavy Chains of Inter Alpha Inhibitor (IαI) Inhibit the Human Complement System at Early Stages of the Cascade

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.

Constitutive Expression of Inter-α-inhibitor (IαI) Family Proteins and Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) by Human Amniotic Membrane Epithelial and Stromal Cells Supporting Formation of the Heavy Chain-Hyaluronan (HC-HA) Complex

Recently, we reported HC-HA, a covalent complex formed between heavy chains (HCs) of inter-α-inhibitor (IαI) and hyaluronan (HA) by the catalytic action of tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), is responsible for human amniotic membrane (AM) anti-inflammatory, anti-scarring, and anti-angiogenic actions. At the present time, the only well characterized source of IαI is serum being produced by the liver. This study showed that AM epithelial and stromal cells and stromal matrix all stained positively for HA, HC 1, 2, and 3, bikunin, and TSG-6. TSG-6 mRNA and protein were constitutively expressed by cultured AM epithelial and stromal cells without being up-regulated by TNF. In serum-free conditions, these cells expressed IαI, leading to the formation of HC-HA complex that contained both HC1 and HC2. In contrast, only HC1 was found in the HC-HA complex purified from AM. Local production of IαI, the HC-TSG-6 intermediate complex, and HC-HA were abolished when cells were treated with siRNA to HC1, HC2, bikunin (all of which impair the biosynthesis of IαI), or TSG-6 but not to HC3. Collectively, these results indicate that AM is another tissue in addition to the liver to constitutively produce IαI and that the HC-HA complex made by this tissue is different from that found at inflammatory sites (e.g. in asthma and arthritis) and in the matrix of the cumulus oocyte complex.

Monocyte-to-Macrophage Differentiation: SYNTHESIS AND SECRETION OF A COMPLEX EXTRACELLULAR MATRIX

Although monocyte- and macrophage-derived molecules are known to promote extracellular matrix (ECM) disruption and destabilization, it is less appreciated that they also synthesize molecules contributing to ECM formation, stabilization, and function. We have identified and characterized the synthesis of proteoglycans and related proteins, some not previously known to be associated with macrophages. Proteoglycan extracts of [(35)S]sulfate- and (35)S-trans amino acid-radiolabeled culture media from THP-1 monocytes induced to differentiate by treatment with phorbol myristate acetate revealed three major proteins of ~25, 90, and 100 kDa following chondroitin ABC lyase digestion. The 25-kDa protein was predominant for monocytes, whereas the 90- and 100-kDa proteins were predominant for macrophages. Tandem mass spectrometry identified (i) the 25-kDa core protein as serglycin, (ii) the 90-kDa core protein as inter-α-inhibitor heavy chain 2 (IαIHC2), and (iii) the 100-kDa core as amyloid precursor-like protein 2 (APLP2). Differentiation was also associated with (i) a >500-fold increase in mRNA for TNF-stimulated gene-6, an essential cofactor for heavy chain-mediated matrix stabilization; (ii) a >800-fold increase in mRNA for HAS2, which is responsible for hyaluronan synthesis; and (iii) a 3-fold increase in mRNA for versican, which interacts with hyaluronan. Biochemical evidence is also presented for an IαIHC2-APLP2 complex, and immunohistochemical staining of human atherosclerotic lesions demonstrates similar staining patterns for APLP2 and IαIHC2 with macrophages, whereas serglycin localizes to the underlying glycosaminoglycan-rich region. These findings indicate that macrophages synthesize many of the molecules participating in ECM formation and function, suggesting a novel role for these molecules in the differentiation of macrophages in the development of atherosclerosis.

Long Pentraxin 3/Tumor Necrosis Factor-Stimulated Gene-6 Interaction

Objective—Angiogenesis is regulated by the balance between pro- and antiangiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor 2 (FGF2) is a major proangiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally coexpressed with its ligand tumor necrosis factor-stimulated gene-6 (TSG-6), a secreted glycoprotein that cooperates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis.Methods and Results—Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine subcutaneous Matrigel plug assay, the inhibition being abolished inPtx3-null mice or by TSG-6 treatment in wild-type animals.Conclusion—TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the coexpression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2.

Tumour necrosis factor-stimulated gene (TSG)-6 controls epithelial–mesenchymal transition of proximal tubular epithelial cells

Progressive renal disease is characterized by accumulation of extracellular matrix in the renal cortex. Proximal tubular cells (PTC) may contribute to disease through a process of epithelial–mesenchymal-transition (EMT): phenotypic change, disruption of the tubular basement membrane and migration into the interstitium. Hyaluronan (HA) synthesis and its extracellular organization by hyaladherins affect cell fate in other systems: this study investigated the role of the hyaladherin, tumour necrosis factor-stimulated gene (TSG)-6, in PTC EMT triggered in vitro by transforming growth factor (TGF)β1. TGFβ1 triggered the loss of PTC epithelial phenotype with 60% decreased expression of E-cadherin and 2–3-fold induction of alpha-smooth muscle actin (α-sma). It also increased the expression of TSG-6, HA-synthase-(HAS)2 and the HA-receptor, CD44, to a peak at 8–12 h, remaining elevated thereafter. Immuno-localization of HA demonstrated that unstimulated PTC assembled HA in cables and that treatment with TGFβ1 initiated cable disassembly with formation of dense HA-pericellular coats. Stable knockdown of TSG-6 with short-hairpin-RNA increased E-cadherin and HAS2 expression, produced loose HA-pericellular coats, HA cables were absent and cell migration was slowed. Treatment of transfectants with TGFβ1 did not induce α-sma, alter E-cadherin, pericellular-HA or migration but did induce HAS2. This was dependent on the expression of CD44 and was inhibited by CD44-specific siRNA. In summary, TSG-6 was central to EMT through effects on HA macromolecular structure and through CD44-dependent triggering of cell responses. These findings suggest that controlling the assembly of HA by proximal tubular cells may be a novel approach towards intervention in renal disease.

The Inflammation-associated Protein TSG-6 Cross-links Hyaluronan via Hyaluronan-induced TSG-6 Oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).