Azithromycin attenuates effects of lipopolysaccharide on lung allograft bronchial epithelial cells.

BACKGROUND: The bronchial epithelium is a source of mediators that may play a role in the airway inflammation and remodeling of post-transplant obliterative bronchiolitis (OB). Traditional strategies have failed to have an impact on OB. Recent studies have suggested a role for azithromycin in managing the condition. In this study we aimed to determine the effect of azithromycin on LPS-mediated epithelial release of factors relevant to airway neutrophilia and remodeling in a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts. METHODS: PBECs were established from bronchial brushings of stable lung transplant recipients and treated with lipopolysaccharide (LPS, 0.1, 1 and 10 microg/ml) for 48 hours. Interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF) protein levels were measured by Luminex analyzer. PBECs were then incubated with LPS and azithromycin, and protein levels were again determined. RESULTS: LPS caused a significant increase in IL-8 and GM-CSF at concentrations of 1 and 10 microg/ml, with no effect on VEGF release. Azithromycin caused a significant decrease in the LPS-stimulated IL-8 and GM-CSF release. CONCLUSIONS: LPS upregulates release of IL-8 and GM-CSF from PBECs derived from stable lung allografts. Sub-microbicidal concentrations of azithromycin attenuate this and may, therefore, alleviate infection-driven neutrophilic airway inflammation and remodeling in the allograft airway.

Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients.

Obliterative bronchiolitis (OB), the major cause of chronic lung allograft dysfunction, is characterized by airway neutrophilia, inflammation, and remodeling, with progressive fibroproliferation and obliteration of small airways that ultimately leads to patient death. Statins have potential anti-inflammatory effects and have been demonstrated to confer a survival advantage in lung transplant patients. We postulated that the beneficial effects of simvastatin in lung transplantation are in part due to inhibition of the epithelial production of key mediators of neutrophil chemotaxis, inflammation, and airway remodeling. Our objective was to assess the effect of simvastatin on a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts, with specific reference to airway neutrophilia and remodeling. PBEC cultures were stimulated with IL-17 or transforming growth factor (TGF)-beta, with and without simvastatin. Supernatant levels of factors critical to driving airway neutrophilia and remodeling were measured. IL-17 upregulated IL-8, IL-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and VEGF, whereas TGF-beta increased IL-6, GM-CSF, matrix metalloproteinase (MMP)-2, and MMP-9. Simvastatin attenuated effects of both IL-17 and TGF-beta. We have demonstrated the ability of simvastatin to attenuate release of airway neutrophilic and remodeling mediators and to inhibit their upregulation by TGF-beta and IL-17. These data illustrate the potential of simvastatin to alleviate neutrophilic airway inflammation and remodeling in the transplanted lung and may have additional relevance to other neutrophilic airway conditions, such as chronic obstructive pulmonary disease.

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis.

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPalpha expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPalpha on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.

Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption.

Cartilage destruction in the arthritides is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, and enzymes from the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family mediate cartilage aggrecan loss. Many genes subject to transcriptional control are regulated, at least in part, by modifications to chromatin, including acetylation of histones. The aim of this study was to examine the impact of histone deacetylase (HDAC) inhibitors on the expression of metalloproteinase genes in chondrocytes and to explore the potential of these inhibitors as chondroprotective agents. The effects of HDAC inhibitors on cartilage degradation were assessed using a bovine nasal cartilage explant assay. The expression and activity of metalloproteinases was measured using real-time RT-PCR, western blot, gelatin zymography, and collagenase activity assays using both SW1353 chondrosarcoma cells and primary human chondrocytes. The HDAC inhibitors trichostatin A and sodium butyrate potently inhibit cartilage degradation in an explant assay. These compounds decrease the level of collagenolytic enzymes in explant-conditioned culture medium and also the activation of these enzymes. In cell culture, these effects are explained by the ability of HDAC inhibitors to block the induction of key MMPs (e.g. MMP-1 and MMP-13) by proinflammatory cytokines at both the mRNA and protein levels. The induction of aggrecan-degrading enzymes (e.g. ADAMTS4, ADAMTS5, and ADAMTS9) is also inhibited at the mRNA level. HDAC inhibitors may therefore be novel chondroprotective therapeutic agents in arthritis by virtue of their ability to inhibit the expression of destructive metalloproteinases by chondrocytes.

The effects of cyclosporin on the collagenolytic activity of gingival fibroblasts.

BACKGROUND: The immunosuppressive agent cyclosporin is associated with a number of major side-effects including the development of gingival overgrowth. Although the pathogenesis of cyclosporin-induced gingival overgrowth remains unclear, it has been suggested that the finely regulated balance between extracellular matrix synthesis and degradation may be disturbed, resulting in an accumulation of excess connective tissue components within the gingival tissue. The aim of this study was to investigate the effect of cyclosporin on matrix metalloproteinases (MMP)-1 and tissue inhibitors of MMP (TIMP)-1 expression at the mRNA, protein, and enzyme activity levels. METHODS: Gingival fibroblasts were grown to confluence and then cultured in serum-free medium supplemented with cyclosporin over the concentration range of 0 to 2000 ng/ml. MMP-1 and TIMP-1 mRNA levels in cultures were determined by reverse transcription polymerase chain reaction (RT-PCR), protein levels in whole conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA), and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. Tissue mRNA levels in normal and overgrown gingiva were also determined by RT-PCR. RESULTS: Results indicated that cyclosporin inhibited MMP-1 expression at both the mRNA and protein level in a dose- and time-dependent fashion. The effects on TIMP-1 expression were less clear, cyclosporin inhibiting mRNA expression, but having no effect on TIMP-1 protein levels at any concentration studied. Addition of the drug resulted in reduced levels of collagenolytic activity in the culture medium. MMP-1 mRNA expression was significantly reduced in overgrown compared to normal tissue. CONCLUSIONS: These results add support to the hypothesis that the accumulation of collagen seen in gingival overgrowth can be explained by a cyclosporin-induced inhibition of collagenolytic activity within the gingival tissues.

The role of soluble interleukin (IL)-6 receptor in mediating the effects of IL-6 on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expression by gingival fibroblasts.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine thought to play a role in the tissue destruction that characterizes periodontal disease. IL-6 exerts its cellular effects through a cell-surface receptor which also exists in a soluble form (sIL-6r). This study investigated the effects of IL-6 on matrix metalloproteinase (MMP)-1 activity in gingival fibroblast cultures, specifically determining the role of the sIL-6r in mediating these actions. METHODS: Fibroblasts were grown to confluence, washed in Hank's balanced saline solution (HBSS), and then cultured for 72 hours in serum-free medium supplemented with 0.2% bovine serum albumin, 1 microgram/ml Escherichia coli LPS and containing various combinations of IL-6 and its soluble receptor over the concentration range 0 to 1,000 ng/ml. MMP-1 and tissue inhibitor of MMP (TIMP)-1 protein levels in the conditioned medium were assessed by enzyme-linked immunosorbent assay (ELISA) and collagenolytic activity determined using a 3H-acetylated type I collagen degradation assay. RESULTS: Results indicated that the addition of IL-6 alone to cultures, over the concentration range 0 to 1,000 ng/ml, had no significant effect on MMP-1 protein expression. However, addition of IL-6 in combination with its soluble receptor resulted in a statistically significant, dose-dependent upregulation in MMP-1 expression. The IL-6/sIL-6r combination also induced a significant increase in collagenolytic activity in cultures. IL-6 and sIL-6r, either alone or in combination, had no marked effect on TIMP expression or cell growth. CONCLUSIONS: These data strongly suggest that future clinical studies investigating the role of IL-6 in periodontal disease must also determine the levels of sIL-6r within the periodontal tissues.