Cytosolic phospholipase A 2a (cPLA 2 ) plays an important role in the development of several inflammatory diseases. The aim of the present study is to determine whether inhibition of cPLA 2 expression, using specific antisense oligonucleotides against cPLA 2 (antisense), is efficient in reducing inflammation after its development. Two mouse models of inflammation were included in the study: thioglicolate peritonitis and collagen-induced arthritis (CIA). The antisense was found to be specific and efficient in inhibiting cPLA 2 expression and NADPH oxidase activity ex vivo in peritoneal phagocytes. Immunoblotting and immunohistochemistry analysis showed a significant elevation in cPLA 2 expression in the inflamed joints of collagen-induced arthritis mice localized in cell infiltrate, chondrocytes and the surrounding skin and skeletal muscle. Similarly, the cPLA 2 metabolite, leukotriene B 4 , accumulated in the peritoneal cavity of mice with peritonitis. Inhibition of elevated cPLA 2 expression after development of inflammation by intravenous administration of antisense resulted in a dramatic reduction in inflammation and a significant reduction in neutrophils recruitment to the site of inflammation in both mouse models of inflammation. Our results demonstrate the critical role of cPLA 2 for the duration of inflammation and suggest that inhibition of cPLA 2 expression by antisense oligonucleotides may serve as an efficient treatment of inflammatory diseases. IntroductionRheumatoid arthritis is an autoimmune inflammatory disease that affects 1% of the population. It is characterized by polyarticular joint inflammation with leukocytic recruitment into synovial fluid and tissue, which appears as a symmetric polyarticular arthritis and which is associated with joint deformities of the hands and feet [1]. Despite extensive efforts, its etiology and pathogenesis remain poorly understood, and effective therapies with limited side effects are still lacking. Collagen-induced arthritis (CIA) in mice is an experimental model that reproduces many of the pathogenic mechanisms of human rheumatoid arthritis, such as increased cellular infiltration, synovial hyperplasia, pannus formation and erosion of cartilage and bone in the distal joints [2]. In humans and in murine arthritis models, chemokines and other chemoattractants [11] knock-out mice suggest that both prostaglandins and LT play an important role in the onset of CIA. Furthermore, dual inhibitors of 5-lipoxygenase and COX were more effective than selective COX inhibitors in preventing arthritis [12]. We have previously provided evidence that cPLA 2 , in addition to its known role in participating in eicosanoid production [13], regulates the phagocyte NADPH oxidase-releasing superoxides [14][15][16]. Moreover, consistent with many other studies, we have demonstrated in vivo and in vitro that during inflammation, the level and activity of both cPLA 2 and NADPH oxidase enzymes are elevated in neutrophils and monocytes [17,18]. Inhibition of overexpressed cPLA 2 protein du...

We have previously demonstrated a physical interaction between cytosolic phospholipase A 2 ␣ (cPLA 2 ) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67 phox or p47 phox , that cPLA 2 specifically binds to p47 phox and that p47 phox is sufficient to anchor cPLA 2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA 2 and p47 phox demonstrated that the cPLA 2 -C2 domain and the p47 phox -PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA 2 -C2 domain mutations were designed to explore its interaction p47 phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA 2 -C2 domain caused a slight inhibition of the affinity binding to p47 phox , whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47 phox -PX domain caused a significant inhibition of the affinity binding to cPLA 2 . Thus, Ile 67 of the cPLA 2 -C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47 phox -PX domain.