Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E2 (PGE2) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE2. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression. METHODS: Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1ß-activated fibroblast-like synoviocytes (FLS) treated with methotrexate. RESULTS: 15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE2 pathway unaltered both in biopsies ex vivo and in cultured FLS. CONCLUSIONS: Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE2 synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE2 synthesis may find a rationale in additionally reducing local inflammatory mediators.

Prostaglandin E2 synthesizing enzymes in rheumatoid arthritis B cells and the effects of B cell depleting therapy on enzyme expression.

INTRODUCTION: B cells may play an important role in promoting immune activation in the rheumatoid synovium and can produce prostaglandin E(2) (PGE(2)) when activated. In its turn, PGE(2) formed by cyclooxygenase (COX) and microsomal prostaglandin E(2) synthase 1 (MPGES1) contributes to the rheumatoid arthritis (RA) pathological process. Therapeutic depletion of B cells results in important improvement in controlling disease activity in rheumatoid patients. Therefore we investigated the expression of PGE(2) pathway enzymes in RA B cells and evaluated the effects of B cell depleting therapy on their expression in RA tissue. METHODS: B cells expressing MPGES1 and COX-2 were identified by flow cytometry in in vitro stimulated and control mononuclear cells isolated from synovial fluid and peripheral blood of RA patients. Synovial biopsies were obtained from 24 RA patients before and at two consecutive time points after rituximab therapy. Expression of MPGES1, COX-1 and COX-2, as well as interleukin (IL)-1ß and IL-6, known inducers of MPGES1, was quantified in immunostained biopsy sections using computerized image analysis. RESULTS: Expression of MPGES1 or COX-2 was significantly upregulated upon stimulation of B cells from blood and synovial fluid while control cells displayed no detectable enzymes. In synovial biopsy sections, the expression of MPGES1, COX-1 or COX-2 was resistant to rituximab therapy at 8 or 16 weeks after start of treatment. Furthermore expression of IL-1ß in the synovial tissue remained unchanged, while IL-6 tended to decrease after therapy. CONCLUSIONS: Therapy with B cell depleting agents, although efficient in achieving good clinical and radiographic response in RA patients, leaves important inflammatory pathways in the rheumatoid synovium essentially unaffected.

Expression of 5-lipoxygenase and 15-lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids.

INTRODUCTION: It was previously shown that lipoxygenase (LO) pathways are important in the rheumatoid arthritis (RA) inflammatory process and that synovial fluid from RA patients contains high amounts of leukotrienes. We therefore aimed to investigate the 5-LO and 15-LO-1 expression pattern in RA and ostheoarthritis (OA) synovial tissue and to study the effect of intraarticular glucocorticoid (GC) therapy on enzyme expression. METHODS: Expression of LOs was evaluated by immunohistochemistry in RA and OA synovial biopsies. Cellular localization of these enzymes was analyzed by double immunofluorescence. In synovial biopsies from 11 RA patients, 5-LO and 15-LO-1 expression was evaluated before and after triamcinolone hexacetonide knee injection and assessed by image analysis to quantify their expression. We also investigated the presence of 15-LO-1 by immunohistochemistry in synovial fluid (SF) cells as well as their ability to form 15-hydroxyeicosatetraenoic acid (15-HETE) following treatment with arachidonic acid (AA). RESULTS: 5-LO and 15-LO-1 are present in RA and OA synovium, with 5-LO being mostly expressed in lining and sublining macrophages, neutrophils and mast cells and 15-LO-1 mainly in lining macrophages, fibroblasts and sublining endothelial cells. Intraarticular GC treatment resulted in a significant suppression of 5-LO expression, but did not influence the 15-LO-1 enzyme significantly. Also, SF cells express a functional 15-LO-1 and produce 15-HETE when challenged with AA. CONCLUSIONS: These data demonstrate that local therapy with GC decreases 5-LO expression in RA synovium and offer an additional possible mechanism for the efficiency of intraarticular adjuvant therapy in RA.

Structural basis for induced formation of the inflammatory mediator prostaglandin E2.

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH(2) into PGE(2). MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH(2) to PGE(2) after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.