- Top of page
- Materials and methods
- Discussion and conclusions
- Conflict of interest
Roflumilast is an oral, once daily investigational PDE4 inhibitor in advanced clinical development for respiratory diseases, such as COPD (Giembycz, 2005; Rabe et al., 2005; Boswell-Smith and Page, 2006). Previous in vitro and in vivo studies revealed the extensive anti-inflammatory potential of roflumilast (Bundschuh et al., 2001; Hatzelmann and Schudt, 2001; Kumar et al., 2003; Jones et al., 2005; Martorana et al., 2005; Mata et al., 2005; Burgess et al., 2006; Growcott et al., 2006; Wollin et al., 2006). Roflumilast reduces antigen-induced inflammatory cell influx and protein accumulation or lipopolysaccharide (LPS)-induced neutrophil influx in bronchoalveolar lavage fluid of Brown–Norway rats in vivo (Bundschuh et al., 2001; Wollin et al., 2003).
Activation of endothelial cells and leukocytes is a hallmark of inflammation that elicits an increase in leukocyte–endothelial interactions and endothelial permeability. Tissue infiltration of leukocytes is preceded by their recruitment from postcapillary venules occurring as a multistep process, initiated by rolling and followed by endothelial adhesion and emigration. These events are orchestrated by sequential expression of cell adhesion molecules (CAMs) on both leukocytes and endothelial cells (Springer, 1994; Kubes and Kerfoot, 2001). Inhibition of PDE4 suppresses leukocyte–endothelial interactions and downregulates CAMs (Sanz et al., 2002, 2005a). Indeed, intravital videomicroscopy of rat mesenteric postcapillary venules revealed that in vivo rolipram (29 μmol kg−1 i.p.) diminishes LPS-induced rapid (0–60 min) or subacute (4 h) leukocyte rolling, adhesion and emigration. In parallel, microvascular P- and E-selectin expressions are abolished (Sanz et al., 2002). In vitro, rolipram reduces neutrophil surface CD11b/CD18 (αMβ2), (Derian et al., 1995; Berends et al., 1997; Sato et al., 2002), tumour necrosis factor-α (TNFα)-induced E-selectin on endothelial cells (Morandini et al., 1996; Blease et al., 1998) or neutrophil adhesion to endothelial cells (Derian et al., 1995; Blease et al., 1998; Jones et al., 2005). Furthermore, enhanced microvascular permeability caused by endothelial cell activation is reversed by cAMP and PDE4 inhibitors (Ortiz et al., 1993; Raeburn et al., 1994; Suttorp et al., 1996).
The present paper describes dose-dependent effects of the PDE4 inhibitor roflumilast on leukocyte rolling, adhesion and emigration at 4 h after stimulation with LPS in rat mesenteric postcapillary venules in vivo, using intravital videomicroscopy (Harris et al., 1994; Johnston et al., 1997; Kubes and Kerfoot, 2001; Sanz et al., 2005a, 2005b). In parallel, the potency and efficacy of roflumilast to inhibit histamine-induced microvascular permeability in rat mesenteric microcirculation was addressed. These in vivo studies were complemented by in vitro investigations exploring direct effects of PDE4 inhibitors, in particular roflumilast N-oxide, on endothelial cells (E-selectin expression, permeability), neutrophils (surface CD11b expression) and neutrophil adherence to endothelial cells. Roflumilast N-oxide is the active metabolite that largely determines the pharmacodynamic activity of roflumilast in rats and in humans (Hatzelmann and Schudt, 2001; Bethke et al., 2007). Our results support the conclusion that roflumilast decreased endothelial cell and leukocyte activation both in vivo and in vitro.
Discussion and conclusions
- Top of page
- Materials and methods
- Discussion and conclusions
- Conflict of interest
The PDE4 inhibitor roflumilast dose-dependently reduced LPS-induced leukocyte–endothelial interactions in rat mesenteric postcapillary venules in a 4-h protocol. In addition, roflumilast suppressed histamine-induced permeability in rat mesenteric microvasculature. Complementary in vitro studies showed that roflumilast N-oxide directly reduced PMNL adherence to HUVEC, neutrophil surface CD11b expression, HUVEC E-selectin expression and macromolecule permeability. Thus, roflumilast decreased endothelial cell activation in vivo and in vitro.
Roflumilast at 10 μmol kg−1 almost completely suppressed LPS-induced leukocyte–endothelial cell interactions in vivo. LPS-induced leukocyte adhesion and emigration were potently reversed by roflumilast. Indeed, extrapolations from pharmacokinetic investigations with roflumilast in Sprague–Dawley rats (data not shown) indicate that at the calculated ID50 values for inhibition of leukocyte adhesion and emigration by the PDE4 inhibitor, free plasma concentrations of roflumilast and roflumilast N-oxide may approach 2–8 nM over the 4-h experimental period corresponding to 50–80% inhibition of PDE4 (Hatzelmann and Schudt, 2001). Therefore, the potency of roflumilast to reduce LPS-induced leukocyte adhesion and emigration in vivo paralleled its capacity to inhibit PDE4. As firm adhesion is mainly governed by leukocyte β2-integrins, it is likely that the potent inhibition of neutrophil surface CD11b upregulation by roflumilast N-oxide in vitro contributed to the strong reduction of LPS-induced leukocyte adhesion in this in vivo model. In fact, in animals pretreated with roflumilast at 10 μmol kg−1, LPS-induced increase of CD11b neutrophil expression was reduced by 47%. On the other hand, leukocyte rolling is governed by a number of CAMs, such as P-/E-selectin, α4-integrin and L-selectin (Johnston et al., 1997; Ley et al., 1998), which may be differentially affected by PDE4 inhibitors. While endothelial P-/E-selectin or neutrophil α4-integrin are reduced (Blease et al., 1998; Sanz et al., 2002; Sullivan et al., 2004 and this study), neutrophil L-selectin may be augmented (Berends et al., 1997). These findings, together with the observation on the complete inhibition of LPS-induced rolling by either L-selectin or α4-integrin antibodies (in the absence of P-selectin) (Johnston et al., 1997), may explain the reduced potency but unchanged efficacy of roflumilast to reduce LPS-induced leukocyte rolling.
Strikingly, roflumilast diminished LPS-induced plasma TNFα increase in Sprague–Dawley rats with approximately the same potency (Bundschuh et al., 2001) as observed for inhibition of LPS-induced leukocyte adhesion and emigration in the current study. Therefore, part of the effects displayed by roflumilast may be due to its rapid inhibition of LPS-induced increase of plasma TNFα in vivo.
In rodents, PDE4 inhibitors may stimulate the hypothalamic–pituitary–adrenal axis. Thus, an increase in endogenous corticosterone plasma levels may, in part, account for the anti-inflammatory effects of PDE4 inhibitors. In mice, plasma corticosterone rose by approximately four- to sixfold after 30 min rolipram administration. The reduction of the LPS-induced TNFα release in an ex vivo whole-blood assay or the ovalbumin-induced pulmonary eosinophilic infiltration by the PDE4 inhibitor was partially reversed by a glucocorticoid receptor antagonist (Pettipher et al., 1997; Kung et al., 2000). In rats, rolipram rapidly (20 min after i.p. injection) and dose-dependently augmented serum corticosterone levels (Kumari et al., 1997). It is therefore possible that the reduction of LPS-induced leukocyte–endothelial cell interactions in the mesenteric postcapillary venules by roflumilast may be attributed, in part, to an increase of serum corticosterone levels. Notwithstanding that, roflumilast and roflumilast N-oxide directly reduced adherence of PMNL to activated HUVEC in vitro.
Given that PDE4 inhibitors attenuate fMLP-, leukotriene B4- or platelet-activating factor-stimulated upregulation of PMNL surface CD11b (Derian et al., 1995; Sato et al., 2002; Meliton et al., 2006 and this study) and considering that endothelial cells activated by cytokines upregulate surface β2-integrin on neutrophils (Kuijpers et al., 1991; Simon et al., 2000), it is proposed that roflumilast N-oxide prevented adherence of resting PMNL to activated endothelial cells by inhibiting the upregulation of PMNL surface β2-integrin. In agreement with this, roflumilast N-oxide or rolipram reduced fMLP-induced PMNL adherence to resting HUVEC with comparable potency and efficacy (Jones et al., 2005 and this study). ADA reversed the inhibition of PMNL adherence to TNFα-activated HUVEC by roflumilast N-oxide and compromised the potency of the PDE4 inhibitor to reduce fMLP-induced surface CD11b on human neutrophils in agreement with earlier studies (Derian et al., 1995; Wollner et al., 1993). Endothelial cells and neutrophils by themselves are strong producers of adenosine that may reinforce the effects of PDE4 inhibitors (Sullivan et al., 2001). These observations suggest that the capacity of roflumilast N-oxide to efficiently reduce neutrophil β2-integrin expression or adherence to endothelial cells may be confined to areas of (neutrophilic) inflammation where adenosine concentrations are reported to be high (10–100 μM) (Hasko and Cronstein, 2004). In contrast, in non-inflamed areas where local adenosine concentrations are low (<1 μM) (Hasko and Cronstein, 2004), the PDE4 inhibitor may be less potent.
Neither roflumilast nor roflumilast N-oxide (up to 1 μM) affected baseline adherence of resting PMNL to unstimulated HUVEC (data not shown). It was shown recently that incubation of HUVEC with roflumilast over 24 h augments baseline IL-8 release, indicating that the PDE4 inhibitor may activate HUVEC. However, this increased IL-8 release was not observed with roflumilast up to 1 μM (that is at concentrations selectively inhibiting PDE4) but occurred at 10 and 100 μM of the compound (McCluskie et al., 2006). At these high concentrations (that is 5000-and 50 000-fold higher than therapeutic plasma levels of roflumilast and roflumilast N-oxide in humans; Bethke et al., 2007), the compound loses its selectivity as an inhibitor of PDE4.
In agreement with previous findings (Pober et al., 1993; Morandini et al., 1996; Blease et al., 1998), dual-selective inhibition of PDE4 and PDE3 reduced HUVEC E-selectin expression by approximately 80%. Motapizone (10 μM), with only approximately 20% inhibition by itself, was synergistic in combination with 1 μM roflumilast N-oxide, where the latter did not affect E-selectin mRNA or protein on its own, reflecting the co-expression of PDE3 and PDE4 in HUVEC (Seybold et al., 2005).
In inflammation, a myriad of mediators directly or indirectly foster endothelial permeability and consequently extravasation of fluids and protein into the extravascular compartment. It is well known that cAMP protects the integrity of the endothelial barrier that is impaired in the presence of thrombin. Recent studies have shown that both protein kinase A and the ‘exchange protein directly activated by cAMP’ (Epac) are critical components in the reduction of endothelial permeability by cAMP (Cullere et al., 2005; Fukuhara et al., 2005; Kooistra et al., 2005; Birukova et al., 2007). Epac-1/Rap1 but also protein kinase A activate Rac, crucial to the provision of an array of cytoskeletal effectors, finally resulting in an improved endothelial barrier. Other mechanisms such as protein kinase A-dependent myosin light chain kinase (MLCK) phosphorylation and RhoA inactivation are also involved in cAMP-dependent protection of the endothelial barrier (Birukova et al., 2007). Consequently, previous investigations have shown that the PDE4 inhibitors rolipram or piclamilast reduce histamine-induced microvascular leakage in guinea pig airways (Ortiz et al., 1993; Raeburn et al., 1994). In our study, roflumilast efficiently suppressed histamine-induced rat mesenteric microvascular permeability in vivo. In fact, among all the in vivo functions explored in this study, microvascular permeability exhibited the highest sensitivity for inhibition by roflumilast. In vitro, roflumilast N-oxide potently diminished thrombin-induced endothelial permeability. The ability of PDE4 and PDE3 inhibitors to protect endothelial barrier integrity in vitro is broadly corroborated by previous studies (Suttorp et al., 1993, 1996; Draijer et al., 1995). It is possible that promoting endothelial barrier integrity by PDE4 inhibitors may potentially contribute to reducing airway oedema in asthma, alveolar oedema in acute lung injury or to mitigating vascular remodelling.
The potencies (IC50) of roflumilast N-oxide or roflumilast compared to rolipram or cilomilast to reduce adherence of PMNL to HUVEC, neutrophil surface CD11b, E-selectin expression and HUVEC permeability are summarized in Table 2 and for comparison, the IC50 for inhibition of PDE4 catalytic activity from human neutrophil extracts (Hatzelmann and Schudt, 2001) are given. For CD11b, IC50 values in the absence of plasma proteins, as shown in Table 2, were estimated from those obtained in the whole-blood assay, considering the following fractions unbound to human plasma: roflumilast 1.1%, roflumilast N-oxide 3.4% (Hauns et al., 2006), rolipram 22%, cilomilast 6% (Hatzelmann and Schudt, 2001). Roflumilast N-oxide and roflumilast reduced endothelial cell and neutrophil functions with IC50 ∼0.5–6.2 nM, which is comparable to their previously reported potency to inhibit PDE4 as well as inflammatory cell functions (Hatzelmann and Schudt, 2001). In addition, plasma concentrations of roflumilast and roflumilast N-oxide required for inhibition of leukocyte adhesion or endothelial permeability by roflumilast in rats in vivo (estimated from ID50) were in the same range as those inhibiting the corresponding endothelial and neutrophil functions in vitro. Roflumilast and roflumilast N-oxide were more potent than rolipram and cilomilast in affecting the investigated endothelial cell functions and endothelial-PMNL adhesion. Cilomilast was the least potent of the four PDE4 inhibitors tested in our assays. This illustrates the higher capacity of roflumilast and its active metabolite to reduce PDE4 activity over rolipram or cilomilast.
In conclusion, by extending earlier investigations that characterized the anti-inflammatory potential of roflumilast, the current study has revealed the capacity of this PDE4 inhibitor to potently suppress the leukocyte–endothelial cell interactions and the increased endothelial permeability that are hallmarks of chronic inflammation in vivo and in vitro.