Pharmacological characterisation of PDE3 and pde4 in human monocytes and monocyte derived macrophages

Parmjit Bahra, Alma Hodzic, Liz Hardaker, Chris Poll, Kathy Banner

Novartis Institutes for Biomedical Research, Horsham, West Sussex, UK

Macrophages have been implicated to play a major role in the pathogenesis of COPD. The lung inflammation associated with this disease is largely insensitive to corticosteroid treatment thus approaches such as those that target cAMP specific phosphodiesterases (PDE’s) has been of considerable interest. There are currently 11 different families of PDE isozymes. The distinct cellular localisation and biophysical characteristics of the various PDE’s suggest that the PDE’s are individually regulated and play distinct roles in specific physiological processes. Elevation of cAMP by PDE3 and PDE4 inhibitors has been shown to inhibit inflammatory mediator release from a number of cell types including monocytes and macrophages (Schudt et.al., 1995).

The aim of this study was to compare the PDE3/4 isozyme gene expression in monocytes and monocyte derived macrophages (MDMs) and pharmacologically characterise the inhibition of LPS induced TNFα release using inhibitors of PDE3, PDE4 and dual PDE3/4 inhibitors.

Blood monocytes were isolated from human donor whole blood using monocyte isolation kit II (Miltenyi Biotech). MDMs were generated through culturing the monocytes in RPMI 1640, 10% FBS, L-glutamine + 2ng/ml GMCSF for up to 12 days. GAPDH, PDE3(A,B), and PDE4(A,B,C,D) mRNA levels were measured by TaqMan RT-PCR (Applied Biosystems). The PDE3 and PDE4 selective inhibitors, trequensin and roflumilast (Sigma) respectively and the dual PDE3/4 inhibitor, SD ISQ 844 (Horikoshi et.al., 1997) were incubated with the monocytes or MDMs for 10 min prior to stimulation with 100ng/ml LPS (E.coli, 026:B6). After 24 hrs the cell free supernatants were assessed for TNFα release by ELISA.

In monocytes the mRNA for all the PDE4 isozymes except for PDE4C was detected. In contrast all of the PDE4 isozymes including low levels of PDE4C were detected in MDMs. The PDE4A isozyme showed the greatest level of expression in both monocytes and MDMs. PDE3B was the only PDE3 isozyme detected in both monocytes and MDMs. These results suggest that the expression profiles of the PDE3 and PDE4 do not greatly alter during the differentiation of monocytes to MDMs.

In monocytes, the PDE4 selective inhibitor, roflumilast, concentration dependently inhibited LPS induced TNFα release with an IC50 (nM) value of 7.0 ± 3.0 (n=7). In contrast roflumilast did not inhibit TNFα release in MDMs. At concentrations where trequensin was selective for PDE3, TNFα release was not affected in either monocytes or MDMs. In contrast, the dual PDE3/4 inhibitor, SDZ ISQ 844 inhibited TNFα release in both monocytes and MDMs. To confirm these findings the effect of roflumilast was tested in the presence of the PDE3 inhibitor trequensin (10nM). It was found that roflumilast concentration dependently inhibited TNFα release from MDMs with an IC50 (nM) value of 30 ± 17(n=4) in the presence of trequensin.

The results of this study demonstrate that differentiation of monocytes to MDMs results in an altered sensitivity to PDE3 and PDE4 inhibitors; however, this is not due to a change in mRNA expression levels. Since macrophages are associated with the pathogenesis of COPD dual PDE3 and PDE4 inhibitors may offer a greater therapeutic advantage over PDE4 inhibitors alone.

Horikoshi S. et.al., (1997). Arerugi, 46(5), 426-432

Schudt C. et.al., (1995). Eur Respir J. 8, 1179-1183