Abstract
Annexin I is a glucocorticoid-induced mediator with anti-inflammatory activity in animal models of arthritis. We studied the effects of a bioactive annexin I peptide, ac 2-26, dexamethasone (DEX), and interleukin-1beta (IL-1beta) on phospholipase A2 (PLA2) and cyclooxygenase (COX) activities and prostaglandin E2 (PGE2) release in cultured human fibroblast-like synoviocytes (FLS). Annexin I binding sites on human osteoarthritic (OA) FLS were detected by ligand binding flow cytometry. PLA2 activity was measured using 3H-arachidonic acid release, PGE2 release and COX activity by ELISA, and COX2 content by flow cytometry. Annexin I binding sites were present on human OA FLS. Annexin I peptide ac 2-26 exerted a significant concentration-dependent inhibition of FLS constitutive PLA2 activity, which was reversed by IL-1beta. In contrast, DEX inhibited IL-1beta-induced PLA2 activity but not constitutive activity. DEX but not annexin I peptide inhibited IL-1beta-induced PGE2 release. COX activity and COX2 expression were significantly increased by IL-1beta. Annexin I peptide demonstrated no inhibition of constitutive or IL-1beta-induced COX activity. DEX exerted a concentration-dependent inhibition of IL-1beta-induced but not constitutive COX activity. Uncoupling of inhibition of PLA2 and COX by annexin I and DEX support the hypothesis that COX is rate-limiting for PGE2 synthesis in FLS. The effect of annexin I but not DEX on constitutive PLA2 activity suggests a glucocorticoid-independent role for annexin I in autoregulation of arachidonic acid production. The lack of effect of annexin I on cytokine-induced PGE2 production suggests PGE2-independent mechanisms for the anti-inflammatory effects of annexin I in vivo.
Highlights
Prostaglandins, such as prostaglandin E2 (PGE2), mediate the pain and edema associated with arthritis, leading to the widespread use of non-steroidal antiinflammatory drugs in the treatment of arthritis
Results demonstrated concentration-dependent annexin I binding, with more than 99% of cells demonstrating annexin I binding at 10 mM (Fig. 1, Table 1)
The hypothesis that annexin I is an anti-inflammatory mediator in arthritis originated from the detection of annexin I in human rheumatoid synovium.[24]
Summary
Prostaglandins, such as prostaglandin E2 (PGE2), mediate the pain and edema associated with arthritis, leading to the widespread use of non-steroidal antiinflammatory drugs in the treatment of arthritis. Within the arthritic synovial lesion, fibroblast-like synoviocytes (FLS) have been implicated as a primary source of PGE2.1 In eicosanoid generation, phospholipase A2 (PLA2) and cyclooxygenase (COX, or prostaglandin H synthase) have been described as important regulatory enzymes. The hydrolytic release of arachidonic acid from membrane phospholipids is initiated by PLA2, and arachidonic acid is catalysed by COX enzymes for the subsequent production of PGE2. PLA2 exists as a secretory group II isoform (sPLA2) and an arachidonic acid-selective cytosolic form (cPLA2).[2] Two isoforms of COX have been identified: a constitutive (COX1) and a mitogen/. Growth factor-inducible (COX2) form.[3] Studies in a COX2 gene knock-out animal model demonstrate that joint inflammation still persists in the absence of this enzyme.[4] At present, the enzymatic events responsible for the enhanced production of PGE2 in rheumatoid arthritis (RA) are not fully understood. It is known that expression of the cPLA2 and COX2 are increased by interleukin-1b (IL-1b)[5,6] and that this induction is inhibited by the anti-inflammatory action of dexamethasone (DEX).[3,6,7]
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