To date, 11 different families of cyclic nucleotide-specific phosphodiesterases (PDEs) have been discovered. These families differ with respect to their regulation and their specificity for either cGMP or cAMP. Of these families, the cAMP-specific PDE, PDE4, is the predominant PDE that regulates inflammatory cell function. The PDE4 family is composed of four genes, PDE4A, 4B, 4C and 4D, and further variants are generated by N-terminal splicing (Houslay et al., 1998). Previous studies of our own have shown that PDE4 is the major isoform regulating the response of basophils (Peachell et al., 1992; Weston et al., 1997; Eskandari et al., 2004). The aim of the present study was to characterise PDE4 isoforms in basophils further. This was achieved by determining whether PDE4 isoforms (PDE4A, 4B, 4C, and 4D) are expressed by basophils using RT-PCR and immunoblotting methodologies.

Basophils were purified (95-100% purity) by immunomagnetic bead separations according to methods that have been described (Weston, et al., 1997). RNA was extracted from the cells using Trizol. Reverse transcription was performed using the Moloney Murine Leukaemia Virus (MMLV) reverse transcriptase. PCR was performed using primers and conditions designed to identify cDNAs for, PDE4A, 4B, 4C and both “short” (4D1/4D2) and “long” forms (4D3, 4D5, 4D7-9) of PDE4D (Seybold et al., 1998; Barber et al., 2004). As a positive control in these PCR experiments, RNA from mononuclear cells (MNC) was used. On occasion, DNA from lung tissue was also used as a positive control. Also, to confirm the integrity of RNA and to gain an estimate of the level of PDE expression, we performed RT-PCR of the house-keeping gene, GADPH. Moreover, Western blots were performed on purified preparations of basophils (95) using antibodies for PDE4A, 4B, 4C, and 4D with recombinant proteins used as positive controls in the blots.

RT-PCR experiments indicate that basophils (n=4-6) express significant levels of message for isoform PDE4A, both “short” and “long” forms of PDE4D with some modest expression of PDE4B but no message for PDE4C. Immunoblotting studies indicate that basophils (n=4) express PDE4A and PDE4D, but no detectable protein for PDE4B or PDE4C.

To conclude, these experiments indicate that PDE4A and PDE4D are the predominant PDE4 subclasses expressed by basophils.

Barber, R. et al. (2004). Am. J. Mol. Physiol. 87, 32-43.
Eskandari, N. et al. (2004). Br. J. Pharmacol. 142, 1265-1272.
Houslay, M.D. et al. (1998). Adv. Pharmacol. 44, 225-342.
Peachell, P.T. et al. (1992). J. Immunol. 148, 2503-2510.
Seybold, J. et al. (1998). J. Biol.Chem. 273, 20575-20588.
Weston, M.C. et al.(1997). Br. J. Pharmacol. 121, 287-295.