Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNFα generation from human monocytes by interacting with a ‘low‐affinity’ phosphodiesterase 4 conformer

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Abstract
1 We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP-specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2-induced cyclic AMP accumulation and lipopolysaccharide (LPS)-induced TNFα production and TNFα mRNA expression. 2 PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP-inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT-PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3 RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 ± 0.6 nM, n = 3). (±)-Rolipram (IC50: 313 ± 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R-(−)−rolipram was approximately 3 fold more potent than S-(+)-rolipram against cytosolic PDE4. 4 RP 73401 (IC50: 9.2 ± 2.1 nM, n = 6) was over 50 fold more potent than (±)-rolipram (IC50: 503 ± 134 nM, n = 6)) in potentiating PGE2-induced cyclic AMP accumulation. R-(−)−rolipram (IC50: 289 ± 121 nM, n = 5) was 4.7 fold more potent than its S-(+)-enantiomer (IC50: 1356 ± 314 nM, n = 5). A strong and highly-significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2-induced monocyte cyclic AMP accumulation and their abilities to displace [3H]-rolipram binding to brain membranes. 5 RP 73401 (IC50: 6.9 ± 3.3 nM, n = 5) was 71 fold more potent than (±)-rolipram (IC50: 490 ± 260 nM, n = 4) in inhibiting LPS-induced TNFα release from monocytes. R-(−)−rolipram (IC50: 397 ± 178 nM, n = 3) was 5.2-fold more potent than its S-(+)- enantiomer (IC50: 2067 ± 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNFα with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]-rolipram binding (r = 0.65, P < 0.01, n = 13). 6 RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml−1)-induced TNFα mRNA. 7 The results demonstrate that RP 73401 is a very potent inhibitor of TNFα release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over-production of this pro-inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]-rolipram from its high-affinity binding site, suggesting that the native PDE4 in human monocytes exists predominantly in a ‘low-affinity’ state.

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