Extended report Ann Rheum Dis 2011;70:1130–1137. doi:10.1136/ard.2010.134825 1130 Accepted 23 January 2011 Published Online First 21 February 2011 ABSTRACT Background The cAMP-metabolising enzyme, phosphodiesterase 4 (PDE4), has been implicated in a number of immune responses, including tumour necrosis factor α (TNFα) production. To date, few data have directly addressed whether synovial cytokine and chemokine production is modified by PDE4. Objective Using specific PDE4 inhibitors, roflumilast plus two novel inhibitors, INH 0061 and INH 0062, the authors studied the effect of PDE4 inhibition on proinflammatory cytokine and chemokine release from primary rheumatoid arthritis (RA) synovial digest suspensions and in a macrophage T cell co-culture assay system. Results All PDE4 inhibitors dose-dependently reduced the release of TNFα from primary synovial membrane cultures (n=5), half maximal inhibitory concentration (IC 50 ) 300–30 nM, p<0.05. Similarly, a significant suppression in the release the proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β (IC 50 300–30 nM) and regulated upon activation normal T-cell expressed and secreted (RANTES) (IC 50 3 nM) was also observed, p<0.05. While interleukin 1β was also reduced, it did not achieve an IC 50 . These observations were further confirmed in a macrophage T cell co-culture system, demonstrating the importance of PDE4 pathways in regulating cytokine/chemokine release in a cellular interaction implicated in inflammatory synovitis. Subsequent studies using the human monocytic cell line U937 also demonstrated cytokine regulation with PDE4 knockdown utilising a small interfering RNA approach. Conclusion These data provide direct evidence of PDE4- dependent pathways in human RA synovial inflammatory cytokine and chemokine release and may provide a novel approach in treating chronic autoimmune conditions such as RA. INTRODUCTION Rheumatoid arthritis (RA) is a chronic, inflamma- tory autoimmune condition primarily affecting the synovial tissues of the joint. RA synovitis is charac- terised by a marked leucocyte infiltration driven in part by local production of proinflammatory cytok- ines and chemokines, which in turn promote ero- sion of cartilage and bone. 1 2 In particular, tumour necrosis factor α (TNFα) has been shown to be pivotal in promoting cytokine and chemokine pro- duction within the RA joint along with cellular acti- vation and articular destruction. 3 Elucidating factors responsible for controlling TNFα and cytokine production is therefore of crucial importance in understanding RA pathogenesis and in identifying new therapies. Given the complexity of the inflammatory cas- cade within the RA joint, there has been increased interest in identifying biochemical signalling moi- eties that might offer intracellular molecular check- points within the cell. 4 In immunocompetent cells, elevation of intracellular cAMP has been proposed as one such moiety. Hydrolysis and degradation of cAMP is specifically controlled by the phosphodi- esterase 4 (PDE4) family of enzymes. Four PDE4 genes have been identified (PDE4A–D), which encode more than 16 different isoforms of the enzyme. 5 Inhibition of PDE4 activity causes elevation of intracellular cAMP levels and subsequent down- regulation of a variety of inflammatory cell activi- ties. In vivo, the specific PDE4 inhibitor, rolipram, has been shown to be an effective regulator of inflammation, 6 7 while in vitro studies have dem- onstrated that specific PDE4 inhibition results in defective effector T cells functions. 8 –12 In addition, further anti-inflammatory effects of PDE4 inhibitors have been reported in a variety of cell lineages. 13 –17 These data provide plausible support for targeting of PDE4 in RA and as such, PDE4 inhibition may offer an attractive option for therapeutic interven- tion in inflammatory diseases. 18 While one study has shown that elevation of cAMP in RA synovial membrane results in reduced TNF release with lit- tle effect on interleukin (IL)-10, 19 crucially there is a paucity of data showing regulation of proinflamma- tory mediators from primary synovial membrane cultures with specific PDE4 inhibitors. Moreover, in human studies thus far undertaken, lipopolysac- charide (LPS) or Staphylococcal enterotoxin B (SEB) has usually been used as the stimulus to study TNFα modulation. 13 14 19 Little information is therefore available on the primary role of PDE4 in spontane- ous cytokine and chemokine release in pathological tissues. Using the specific PDE4 inhibitor roflu- milast and two novel PDE4 inhibitors, INH0061 and INH0062, we have addressed this question directly in primary RA synovial digest suspension cultures. Briefly, INH0061 and INH0062 are two recently generated PDE4 inhibitors, both of which are quinoline-derived compounds. 20 INH 0061 is a potent, reversible inhibitor of the PDE4B enzyme and shows >9000-fold selectivity over PDE1, -2, -3, -5, -6 and -7 and all other off-target enzymes and receptors tested. In human whole blood, INH 0061 inhibits LPS-stimulated production of TNFα with an IC 50 of 7.6 nM. Similarly, INH 0062 is a 1 Institute of Infection, Immunity and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK 2 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 3 GlaxoSmithKline Medical Research Centre, Stevenage, UK Correspondence to Dr Anne Crilly, Level 4, McGregor Building, Western Infirmary, Glasgow G11 6NT, UK; anne.crilly@glasgow.ac.uk Phosphodiesterase 4 (PDE4) regulation of proinflammatory cytokine and chemokine release from rheumatoid synovial membrane Anne Crilly, 1 Susan E Robertson, 1 James H Reilly, 1 J Alastair Gracie, 1 Wen-Qi Lai, 2 Bernard P Leung, 1,2 Paul F Life, 3 Iain B McInnes 1 group.bmj.com on December 18, 2013 - Published by ard.bmj.com Downloaded from