IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-B, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.Because of its diverse biological function and simultaneous description in different studies, IL-6 was initially assigned several names. It was identified as a T-cell-derived B-cell differentiation factor, as it induced activated B-cells into antibody-producing cells: interferon-␤2 (26 kDa protein), a hybridoma/plasmacytoma growth factor and a hepatocytestimulating factor. The name IL-6 was proposed when the cDNA nucleotide sequences for these proteins had been determined, and the molecules were found to be identical (1). In addition, IL-6 plays a key role in inflammation, being the main inducer of fibrinogen, serum amyloid A protein, the acute phase response and is one of the most important mediators of fever. In muscle and fatty tissues, IL-6 stimulates energy mobilization.The IL-6 promoter is rapidly activated by cytokines, including IL-1 and TNF-␣, as well as by phorbol esters and cyclic AMP. The promoter-region of the IL-6 gene contains multiple regulatory elements such as nuclear factor-B (NF-B), nuclear factor-IL6 (NF-IL6) (also referred to as C/EBP␤), cAMP response element (CRE), TPA (12-O-tetradecanoylphorbol-13-acetate) responsive element (TRE; also referred to as the AP-1 binding site), and the glucocorticoid response element (GRE) (2).Structurally related to TRE is the antioxidant responsive element (ARE, 2 also referred to as the electrophile responsive element (EpRE)) (3, 4). Some TREs are found to be embedded within an ARE, such as in the promoter region of human NAD(P)H:quinine oxidoreductase-1 (NQO1), rat and mouse glutathione S-transferase (GST) Ya subunit, and rat GST-P (5). ARE is commonly found in the promoter region of genes encoding phase II detoxification as well as antioxidant enzymes such as NQO1, thioredoxin, thioredoxin reductase, glutathione peroxidase, and hemeoxygenase-1 (6). Analyses of ARE-nuclear protein complexes have identified numerous nuclear transcription factors including c-Jun, Jun-B, Jun-D, c-Fos, Fra1, Nrf1, Nrf2, ...

Objective. Treatment options for rheumatoid arthritis range from symptomatic approaches to modern molecular interventions such as inhibition of inflammatory mediators. Inhibition of inflammation by plateletrich plasma (PRP) has been proposed as a treatment for tendinitis and osteoarthritis. The present study was undertaken to investigate the effect of PRP on antigeninduced arthritis (AIA) of the knee joint in a large animal model.Methods. Six-month-old pigs (n ‫؍‬ 10) were systemically immunized by bovine serum albumin (BSA) injection, and arthritis was induced by intraarticular BSA injection. PRP was injected into the knee joints of 5 of the animals after 2 weeks. An additional 5 animals received no systemic immunization (controls). Signs of arthritis were documented by plain histologic analysis, Safranin O staining, and immunohistochemistry analysis for type II collagen (CII), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF). Interleukin-1␤ (IL-1␤), IL-6, tumor necrosis factor ␣ (TNF␣), VEGF, and insulin-like growth factor 1 (IGF-1) protein content was measured by Luminex assay.Results. In the pigs with AIA, plain histologic analysis revealed severe arthritic changes in the synovium. Safranin O and CII staining showed decreased proteoglycan and CII content in cartilage. Immunohistochemistry analysis revealed increased levels of IL-6 and VEGF in synovium and cartilage, and protein concentrations of IL-6, VEGF, IL-1␤, and IGF-1 in synovium and cartilage were elevated as well; in addition, TNF␣ protein was increased in cartilage. Treatment with PRP led to attenuation of these arthritic changes in the synovium and cartilage. Conclusion. We have described a porcine model of AIA. Experiments using this model demonstrated that PRP can attenuate arthritic changes as assessed histologically and based on protein synthesis of typical inflammatory mediators in the synovial membrane and cartilage.

Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds.

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/10(9) platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.