Arthritic Cell-Based Assay Model for Measuring Inflammatory Response to Therapeutic Biological Agents




Sohrabi, Afshin

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Rheumatoid arthritis (RA) is the most common inflammatory joint disease with considerable morbidity and mortality. Conventional disease-modifying anti-rheumatic drugs like methotrexate and NSAIDs form the cornerstone of therapy. However, they have several limitations in terms of slow onset of action, adverse effects and modest remission and retention rates. Several cytokines are involved in the pathogenesis of RA. Biological agents that specifically inhibit the effects of tumor necrosis factor-£\ (TNF-£\) or MMP-1 represent a major advancement in the treatment of RA. Here we describe and characterize a novel human synoviocyte-based model for the anti-arthritis drug screening, we found that the treatment of cultured human synoviocytes with LPS induces an inflammatory response. The recombinant anti-£\ trypsin, rAAP, suppresses this response. Using Crystal Violet assays, we showed that rAAP is not cytotoxic to human synoviocytes at up to 10<em>f</em>ÝM concentrations. To explore the anti-inflammatory properties of rAAP in human synoviocytes stimulated with LPS, the levels of inflammatory mediators and an activity of NF-kB/IkB-<em>f</em>Ñ signaling pathway were quantified across various time points post rAAP treatment. Using qRT-PCR, we showed that rAAP inhibits the expression of TNF-alpha, IL-1beta, IL-6, GM-CSF, MMP1, and COX-2. Recombinant AAP suppresses the LPS-induced production of TNF-alpha levels to ~ 30% of that in untreated control cells. By stimulating phosphorylation of the inhibitory IkB-alpha protein at Ser32 and Ser36 followed by proteasome-mediated degradation, rAAP affects NF-kB activation which plays a central role in the inflammatory responses to LPS activation. Since the NF-kB/IkB-alpha signaling pathway is involved in cytokine production, it is also inhibited. Hence, the mechanism of action appears to be through the modulation of proinflammatory mediators such as TNF-alpha, IL-1beta, IL-6, COX-2, MMP1, prostaglandin E2, GM-CSF, and IL-8. These observations suggest that inhibition of TNF-alpha cascade by rAAP may help reduce the inflammation for RA. Our studies indicates that LPS activated human synoviocytes model may help in further investigation of the mechanisms involved in the inflammatory cascade observed in RA.



Molecular biology, Cellular biology, Arthritis, Bilogicals, Cell Based Assay, Gene Expression, Inflammation, Protein Expression