RESULTS

The effects of PTX on LPS-induced TNF-α production
To determine the duration of PDE inhibition on TNF-α production after LPS stimulation, time course studies were conducted. In accordance with prior work, PTX had a rapid and sustained effect on TNF-α production in vitro (Figure 1).

PTX decreases LPS-induced NF-κB activation
Since phosphorylation leads to ubiquitination and degradation of I-κBα and subsequent nuclear translocation of NF-κB, we first examined the effects of PTX on I-κBα. Cytoplasmic I-κBα phosphorylation was markedly increased following LPS stimulation when compared to control (100 ± 0 vs. 20 ± 18; P < 0.05). The addition of PTX significantly downregulated LPS-induced I-κBα phosphorylation (P = 0.02; Figure 2)
In a similar fashion, nuclear NF-κB p65 phosphorylation was increased following LPS stimulation (100 ± 0 vs. 38 ± 20; P < 0.01). PTX similarly decreased LPS-induced NF-κB p65 nuclear phosphorylation, a marker of NF-κB activation and nuclear translocation (100 ± 0 vs. 40 ± 6; P =0.03; Figure 3A).
EMSAs were then performed to verify the PTX-induced alterations in nuclear phosphorylation resulted in similar effects on the DNA binding activity of NF-κB. The addition of PTX to LPS-stimulated mononuclear cells resulted in comparable downregulation of DNA binding activity, similar to the observed downregulation of NF-κB phosphorylation (Figure 3B).

PTX upregulates CREB phosphorylation and activation after LPS stimulation
Phosphorylation of nuclear CREB was used as a marker for CREB activation. LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A). PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS). When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 ± 92 vs. 100 ± 0; P < 0.01). In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).
To determine if CREB-DNA binding was affected by PTX in a manner similar to CREB phosphorylation, an EMSA was performed. Exposure of LPS-stimulated cells to PTX similarly increased CREB DNA-binding compared to LPS alone (Figure 4B).

Effect of PTX on TNF-α production in the presence of PKA inhibition
The role of PKA in PTX-induced downregulation of TNF-α production in LPS-stimulated mononuclear cells was assessed by treating mononuclear cells with H89, a PKA inhibitor, prior to LPS and PTX exposure. The addition PTX to LPS-stimulated cells resulted in a reduction in TNF-α, similar to that observed in our previous experiments (529 pg/mL ± 112 vs. 37 pg/mL ± 10; P < 0.01). Pre-incubation of cells with H89 prior to LPS and PTX exposure did not result in any significant modulation of TNF-α production when compared to that demonstrated with PTX alone (P = 0.2; Figure 5). These results suggest that downregulation of TNF-α production by PTX in LPS-stimulated mononuclear cells is, at least in part, PKA-independent.