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THU0245 Phosphodiesterase 4 Expression in Rheumatoid Arthritis Synovium and Effects of Apremilast on Synovial Fibroblasts
  1. L. Wu1,
  2. M. F. Adams1,
  3. A. Parton1,
  4. P. H. Schafer1
  1. 1Celgene Corporation, Summit, United States

Abstract

Background Apremilast (APR), an oral small-molecule inhibitor of phosphodiesterase 4 (PDE4) enzymes, works intracellularly to modulate a network of pro-inflammatory and anti-inflammatory mediators. PDE4 is a cyclic adenosine monophosphate (cAMP)-specific PDE, with PDE4A, B, and D being the major PDE4 enzymes expressed in leukocytes. PDE4 inhibition elevates intracellular cAMP levels, which in turn down-regulates the inflammatory response by modulating the expression of TNF-α, IL-23, IL-17 and other inflammatory cytokines. However, the expression and function of PDE4 in rheumatoid arthritis (RA) synovium and synovial fibroblasts has not yet been fully elucidated.

Objectives The expression pattern of the major PDE4 enzymes was studied in normal and RA synovium, and in normal and RA synovial fibroblasts (SF). The effects of APR on intracellular cAMP levels and on the production of major destructive proteases by RASF were examined.

Methods PDE4A, B, and D protein expression was measured in whole synovial tissue or isolated synovial fibroblasts from individuals with RA or from normal controls by quantitative laser scanning cytometry (iCyte) and by semiquantitative immunohistochemistry (IHC). Gene expression in normal SF, RASF, peripheral blood mononuclear cells from RA and normal controls was measured by qRT-PCR. RASF cell cultures were treated with 0.1-10 µM APR and then stimulated with 10 ng/mL IL-1β, TNF-α, IL-17, or IL-6 for a total of 24 hours, then supernatants were collected for analysis of matrix metalloproteinase (MMP)1, MMP3, MMP13, and MMP14 by ELISA.

Results Laser scanning cytometry of synovial tissue from RA patients showed higher overall expression of PDE4A, B, and D protein compared to normal synovium (p<0.001). IHC staining also showed higher PDE4A, B, and D protein levels in the superficial synoviocytes and subsynovial histiocytes in RA samples compared to normal controls. Laser scanning cytometry of normal synovial fibroblasts and RASF showed similar strong cytoplasmic staining of PDE4A in normal and RA samples. PDE4B showed abundant cytoplasmic staining, with 45% higher staining in RASF compared with controls (p<0.01). PDE4D showed moderate to strong cytoplasmic staining in normal samples, but 31% weaker expression in RASF samples (p<0.05). qRT-PCR analysis of PDE4A and PDE4B gene expression was similar in RASF and controls, but PDE4D gene expression was 60% lower in RASF compared with controls (p<0.01). In RASF cell cultures, APR treatment increased intracellular cAMP levels. APR significantly inhibited RASF production of all MMPs induced by IL-1β stimulation, whereas in TNF-α-stimulated RASF cultures, APR inhibited MMP3 and MMP14 production. In the IL-17- or IL-6-stimulated cultures, no significant inhibition of MMP production was observed.

Conclusions Overall, PDE4A, B, and D protein expression was stronger in RA vs. normal synovium, largely due to increases in superficial synoviocytes, subsynovial histiocytes, and lymphoplasmacytic cells. In isolated RASF, there is a shift in expression away from PDE4D toward PDE4B expression. In RASF, APR is capable of elevating intracellular cAMP and inhibiting MMP production in response to the JAK-independent stimuli, IL-1β and TNF-α. This study provides the preclinical rationale for using APR in RA.

Disclosure of Interest L. Wu Employee of: Celgene Corporation, M. Adams Employee of: Celgene Corporation, A. Parton Employee of: Celgene Corporation, P. Schafer Employee of: Celgene Corporation

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