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Abstract
Neutrophilic inflammation is characteristic of chronic obstructive pulmonary disease (COPD); yet, there are no effective antiinflammatory therapies. The PDE4 inhibitor roflumilast is approved for use in COPD and suppresses sputum neutrophilia. The mechanism underlying this observation is unclear; therefore, this study addressed whether roflumilast directly affected neutrophil migration. Blood-derived neutrophils were isolated from nonsmokers, smokers, and patients with COPD, and chemotaxis was measured using Boyden chambers. Intracellular calcium ion concentration was measured by fluorimetry, and shape change and CD11b expression were measured by flow cytometry. Neutrophils from patients with COPD showed enhanced chemotactic responses toward both CXCL1 and leukotriene B4 compared with control cells. Chemotaxis was inhibited by both the active metabolite roflumilast N-oxide and rolipram in a concentration-dependent manner with no difference in responsiveness between subjects. Roflumilast N-oxide and rolipram were less efficacious against CXCL1 and leukotriene B4–mediated intracellular calcium ion concentration, suggesting that inhibition was not via this pathway. Both PDE4 inhibitors attenuated chemoattractant-mediated shape change and CD11b upregulation, suggesting common mechanisms. The stable cAMP analog 8-bromoadenosine 3′,5′-cAMP inhibited chemotaxis, as did the direct Epac1 (exchange protein directly activated by cAMP 1) activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cAMP but not the direct protein kinase A activator N6-benzoyladenosine-3′,5′-cAMP. These data suggest that roflumilast inhibits neutrophil chemotaxis directly via a cAMP-mediated mechanism requiring activation of Epac1 and that Epac1 activators could reduce COPD neutrophilic inflammation.
Chronic obstructive pulmonary disease (COPD) is defined as airflow limitation that is not fully reversible and is associated with an underlying pulmonary inflammation (1). It is increasing in prevalence worldwide and is believed to affect approximately 10% of the global population over the age of 40 years (2). The mechanisms underlying the pathology of COPD remain elusive, with no therapeutic interventions available currently that can prevent the progressive decline in lung function seen in these patients (3). Inflammation in COPD is associated with increased neutrophilia in the airways (3) and can be assessed by an increase in neutrophil numbers in sputum (4). This increase in neutrophil numbers can be explained by a concomitant increase in neutrophil chemotactic factors, including CXCL1 (growth-related oncogene-α), CXCL8 (IL-8), and leukotriene B4 (LTB4) (5–7); yet, most antiinflammatory approaches, including glucocorticosteroids, have failed to suppress this abnormal inflammatory response in COPD (8).
More recently, administration of a PDE4 inhibitor, roflumilast, showed benefit in a subsection of patients with COPD by reducing the frequency of exacerbations (9–11), but it may also have additional effects on lung function (12) and inflammatory indices including sputum neutrophilia (13). The mechanisms of this antiinflammatory effect of roflumilast are not clear, although the effect may occur through elevation of intracellular cAMP and suppression of epithelium-derived chemokines (14). Alternatively, roflumilast could be acting directly on neutrophils to suppress function. The latter is consistent with studies that have shown that roflumilast can alter expression of adhesion molecules and inhibits adhesion of human neutrophils to endothelial cells and upregulation of FMLP-stimulated CD11b expression (15). Roflumilast has also been shown to suppress reactive oxygen species production from human neutrophils stimulated with FMLP, murine neutrophil chemotaxis to C5a (16), and human neutrophil chemotaxis to both CXCL8 and LTB4 (17). Moreover, PDE4 inhibition can also suppress human neutrophil degranulation in vitro (18). These data are further supported by a proof-of-concept study in which healthy volunteers were given 28 days of oral roflumilast (500 μg once daily) or matched placebo, followed by segmental endotoxin challenge and bronchoscopy (19). In subjects who had been given the PDE4 inhibitor, there was a significant reduction in the numbers of neutrophils and eosinophils accumulating in the lungs compared with placebo (19). These studies provide support that PDE4 inhibition may affect antiinflammatory responses via suppression of neutrophil migration. This could be important in the context of COPD because there is evidence that neutrophil migration is abnormal in this disease (20–22) and could contribute the destructive pathophysiology observed in these patients.
The mechanism by which PDE4 inhibition could suppress neutrophil migration is believed to be via elevation of cAMP (23, 24) and subsequent activation of target enzymes such as protein kinase A (PKA). Activation of PKA leads to inhibition of cell surface expression of integrins and, via inhibition of RhoA (Ras homolog gene family, member A), to modulation of Ca2+-mediated regulation of the actin cytoskeleton (25). cAMP is also known to activate Epac1 (exchange protein directly activated by cAMP 1) in neutrophils (26), and this stimulates the Ras GTPase homolog Rap1 (26, 27). The expression of Epac2 has not been observed in these cells. Translocation of Rap1 and its effector protein RAPL to the leading edge of the migrating leukocyte leads to upregulation of β2-integrins for increased adhesion and motility and highlights the duality of cAMP activity (25). However, Epac1-dependent activation of Rap1 in human neutrophils fails to increase expression of β2-integrins and therefore may have a differential effect in these cells (26).
The aims of this study were to examine the effect of the active metabolite of roflumilast, roflumilast N-oxide, on neutrophil migration and to compare efficacy between cells from patients with COPD and control subjects. In this study, we also examined the putative mechanisms of action of roflumilast N-oxide using the cAMP analog 8-bromoadenosine 3′,5′-cAMP (8-Br-cAMP) and sought to determine if the mechanism was independent of activation of PKA using the activator N6-benzoyladenosine-3′,5′-cAMP (6-Bnz-cAMP) and the Epac activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP).
Healthy nonsmoking subjects (n = 25), smokers without COPD (n = 13), and patients with COPD (both current and ex-smokers) (n = 20) were recruited from the National Heart and Lung Institute, Royal Brompton Hospital, London, United Kingdom. COPD was defined according to Global Initiative for Chronic Obstructive Lung Disease criteria (1). All subjects gave written informed consent as approved by the London-Chelsea National Research Ethics Committee. Patients with COPD were required to have been free from exacerbation or antibiotic or steroid treatment for the preceding 6 weeks. Demographic data are presented in Table E1 in the data supplement. Patients with COPD were significantly older than subjects in the control groups, but there were no differences in smoking history between patients with COPD and the smoking control subjects (Table E1).
Neutrophils were isolated from whole blood using Percoll density gradients as described previously (28), and migration was evaluated using a 48-well Boyden chamber (28, 29) after incubation in the absence or presence of the active metabolite of roflumilast (roflumilast N-oxide) or rolipram.
Calcium measurements were performed as described previously. Briefly, cells were resuspended at 10 × 106 cells per milliliter in calcium buffer (20 mM HEPES, 138 mM NaCl, 6 mM KCl, 1 mM MgCl2, 5.2 mM glucose, 1 mM CaCl2, 1% [wt/vol] BSA, pH 7.4) and incubated with 4 μM Fura-2AM and 0.02% (vol/vol) pluronic acid in the presence or absence of roflumilast N-oxide, rolipram, or vehicle for 30 minutes at 37°C in the dark. Excess dye was removed by centrifugation, and cells were resuspended at 10 × 106 cells per milliliter in calcium buffer with the appropriate drug or vehicle. Fura-2–loaded cells (1 × 106 cells) were added to each well of a 96-well black plate, and the cells were equilibrated for 10 minutes at 37°C. Cells were stimulated with agonist or calcium buffer. Excitation (340/380 nm) and emission (510 nm) were recorded using a FLUOstar Optima plate reader (BMG LABTECH Ltd.) at 2.8-second intervals. The Ca2+ concentration was calculated using the Grynkiewicz equation (30). Rmax (maximum ratio [R] of fluorescence at the two wavelengths) was obtained using 0.25 mg/ml digitonin, and Rmin was obtained using 25 mM EDTA.
Whole blood from all subjects was stimulated with chemoattractants after incubation with formoterol (1 μM) for 10 minutes at 37°C and rolipram or roflumilast N-oxide for 10 minutes, and shape change was assessed by flow cytometry (31).
RNA was extracted from neutrophils using the RNeasy kit (Qiagen), and TaqMan quantitative reverse transcriptase PCR were assays performed (Epac1, Hs01030417_m1; Epac2, Hs00199754_m1) and normalized to the housekeeping gene HPRT (hypoxanthine-guanine phosphoribosyltransferase; Hs0200695_m1) (Applied Biosystems/Life Technologies).
Cell viability was measured by Trypan blue exclusion. Isolated neutrophils were greater than 99% viable, and none of the drugs altered cell viability.
Data are presented as mean ± SEM for a given number (n) of observations. Differences in n between experiments are due to differences in the yield of neutrophils in each sample. Comparisons between subject groups were performed using either two-way ANOVA followed by Bonferroni correction or Kruskal-Wallis analysis using Prism software (GraphPad Software Inc.), followed by Dunn’s multiple comparisons test or a Mann-Whitney U test when appropriate. Differences were considered significant when P < 0.05.
Initial experiments were performed using neutrophils from patients with COPD and control subjects to determine the concentrations of chemoattractants to use in subsequent experiments. Neutrophils showed a bell-shaped chemotactic response to CXCL1 (Figure 1A) with an increased migration of cells from patients with COPD compared with cells from nonsmokers. Similar experiments were performed to determine the chemotactic response to LTB4, and again a significant increase in the response of neutrophils from patients with COPD to LTB4 was observed (Figure 1B). These data suggested that neutrophils from patients with COPD showed a heightened response to chemotactic stimuli. On the basis of these data, 3 nM CXCL1 and 1.3 nM LTB4 were selected for future chemotaxis experiments. Activation of chemoattractant receptors and subsequent migration is believed to require elevations in intracellular calcium ion concentration ([Ca2+]i), and therefore concentration responses were performed for CXCL1 (Figure 1C) and LTB4 (Figure 1D). On the basis of these experiments, 10 nM CXCL1 and 30 nM LTB4 were selected to examine the effects of PDE4 inhibitors on [Ca2+]i.
Figure 1. Effect of CXCL1 and leukotriene B4 (LTB4) on neutrophil chemotaxis and intracellular calcium ion concentration ([Ca2+]i). (A and B) Neutrophils were isolated from the blood of nonsmokers (solid circles; n = 4), smokers (open circles; n = 4), and patients with chronic obstructive pulmonary disease (COPD) (solid triangles; n = 5) and chemotaxis performed toward either CXCL1 (A) or LTB4 (B). Cells were counted, and data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 for differences between neutrophils from nonsmokers and patients with COPD, and +P < 0.05 and ++P < 0.01 for differences between neutrophils from smokers and patients with COPD using two-way ANOVA followed by Bonferroni correction. (C and D) Typical calcium transients for CXCL1 (C) and LTB4 (D). The black line is basal (no agonist), and the agonist concentrations are represented by different-colored lines as follows: red = 1 nM; orange = 3 nM; green = 10 nM; purple = 30 nM; blue = 100 nM.
[More] [Minimize]The PDE4 inhibitors roflumilast N-oxide and rolipram both inhibited migration of neutrophils toward CXCL1 (3 nM) and LTB4 (1.3 nM) in a concentration-dependent manner (Figure 2). There were no significant differences in the inhibitory effects of either of these PDE4 inhibitors on neutrophils from nonsmokers, smokers, or patients with COPD (Figure 2 and Table 1); however, roflumilast N-oxide was more potent than rolipram at inhibiting migration toward both chemoattractants (Table 1). The chemoattractant did not alter the potency or efficacy of the inhibitors (Table 1). This suggests that neither roflumilast N-oxide nor rolipram was acting at a specific chemoattractant receptor or pathway. Therefore, to investigate where in the chemoattractant signaling pathway the PDE4 inhibitors were exerting their effects, experiments were devised in which measurements of intracellular calcium transients were performed in the presence of both inhibitors.
Figure 2. Effect of roflumilast N-oxide and rolipram on neutrophil chemotaxis toward CXCL1 and LTB4. (A–D) Neutrophils were isolated from the blood of healthy subjects (solid circles; n = 6–10), smokers (open circles; n = 6–9), and patients with COPD (solid triangles; n = 3–7) and preincubated in either roflumilast N-oxide (A and C) or rolipram (B and D) for 1 hour before chemotaxis toward either CXCL1 (3 nM) (A and B) or LTB4 (1.3 nM) (C and D). Cells were counted, and data are presented as mean ± SEM as a percentage of cells migrated in the absence of drug. There is no difference in the responsiveness of neutrophils from the three groups (Kruskal-Wallis test).
[More] [Minimize]| Drug | Chemotaxis (EC50 in nM) | [Ca2+]i (EC50 in nM) | |||
|---|---|---|---|---|---|
| CXCL1 | LTB4 | CXCL1 | LTB4 | ||
| Nonsmokers | Roflumilast N-oxide | 0.40 ± 0.2 | 0.55 ± 0.29 | 70.2 ± 30.9 | 17.8 ± 10.1 |
| n = 10 | n = 6 | n = 8 | n = 5 | ||
| Rolipram | 4.2 ± 1.4 | 3.1 ± 2.5 | 980 ± 253 | 657 ± 424 | |
| n = 10 | n = 6 | n = 7 | n = 5 | ||
| Smokers | Roflumilast N-oxide | 1.5 ± 0.9 | 1.4 ± 1.4 | 52.9 ± 36.9 | 325 ± 322 |
| n = 9 | n = 6 | n = 7 | n = 4 | ||
| Rolipram | 37.0 ± 27.2 | 78 ± 52.5 | 1779 ± 1014 | 2623 ± 1504 | |
| n = 9 | n = 6 | n = 8 | n = 4 | ||
| COPD | Roflumilast N-oxide | 4.8 ± 3.2 | 7.9 ± 3.9 | 39.1 ± 13.7 | NC |
| n = 7 | n = 3 | n = 7 | |||
| Rolipram | 15.8 ± 11.2 | 172 ± 119 | 93.4 ± 59.5 | NC | |
| n = 7 | n = 3 | n = 7 | |||
Both roflumilast N-oxide and rolipram inhibited CXCL1-mediated [Ca2+]i in a concentration-dependent manner (Figure 3). Again, there was no difference in the response of neutrophils from nonsmokers, smokers, or patients with COPD to either of these inhibitors (Figure 3 and Table 1); however, the half-maximal effective concentration (EC50) for the inhibition of chemotaxis and [Ca2+]i differed by at least one order of magnitude (Table 1). In contrast to the effects on CXCL1-mediated [Ca2+]i, both PDE4 inhibitors had limited effect on LTB4-induced [Ca2+]i, with maximum inhibition of the peak response of about 25% by both roflumilast N-oxide and rolipram (Figure 4). This limited inhibition was lost in neutrophils from patients with COPD, suggesting a difference in sensitivity in these cells (Figure 4D and Table 1).
Figure 3. Effect of roflumilast N-oxide and rolipram on neutrophil CXCL1-induced [Ca2+]i. (A–D) Neutrophils were isolated from the blood of healthy subjects (solid circles; n = 6), smokers (open circles; n = 6), and patients with COPD (solid triangles; n = 6) and preincubated in either roflumilast N-oxide (A and C) or rolipram (B and D) for 1 hour before measurements of [Ca2+]i toward CXCL1 (10 nM). (A and B) Typical calcium transients. The black line is CXCL1, and the PDE4 inhibitor concentrations are represented by different-colored lines as follows: red = 10−5 M; orange = 10−6 M; green = 10−7 M; purple = 10−8 M; blue = 10−10 M. (C and D) Data are presented as mean ± SEM as a percentage of the peak calcium response in the absence of drug. There is no difference in the responsiveness of neutrophils from the three groups (Kruskal-Wallis test).
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Figure 4. Effect of roflumilast N-oxide and rolipram on neutrophil LTB4-induced [Ca2+]i. (A–D) Neutrophils were isolated from the blood of healthy subjects (solid circles; n = 5), smokers (open circles; n = 4), and patients with COPD (solid triangles; n = 3) and preincubated in either roflumilast N-oxide (A and C) or rolipram (B and D) for 1 hour before measurements of [Ca2+]i toward LTB4 (30 nM). (A and B) Typical calcium transients. The black line is LTB4, and the PDE4 inhibitor concentrations are represented by different-colored lines as follows: red = 10−5 M; orange = 10−6 M; green = 10−7 M; purple = 10−8 M; blue = 10−10 M. (C and D) Data are presented as mean ± SEM as a percentage of the peak calcium response in the absence of drug. There is no difference in the responsiveness of neutrophils from the three groups (Kruskal-Wallis test).
[More] [Minimize]These data suggested that the effects of PDE4 inhibitors on chemotaxis and [Ca2+]i may occur by differing mechanisms. Therefore, the effects of these inhibitors on surrogate measures of neutrophil activation were also examined. The effects of roflumilast N-oxide and rolipram on CXCL1- and LTB4-mediated shape change and CD11b upregulation in neutrophils from nonsmokers, smokers, and patients with COPD were evaluated using whole-blood assays. However, neither of these inhibitors altered any of the parameters measured (data not shown). It was possible that the lack of effect of PDE4 inhibitors on these responses might be due to low concentrations of intracellular cAMP; therefore, whole blood was pretreated with 1 μM formoterol, a β2-adrenergic receptor agonist, and before exposure to PDE4 inhibitors. Under these conditions, both roflumilast N-oxide and rolipram inhibited CXCL1-mediated neutrophil shape change and CD11b cell surface expression in a concentration-dependent manner (Figure 5 and Table E2). Again, there was no difference in the responses of neutrophils from nonsmokers, smokers, or patients with COPD to these agents. However, the EC50 concentrations of these responses (Table E2) were again higher than those required to elicit an EC50 response for chemotaxis (Table 1).
Figure 5. Effect of roflumilast N-oxide and rolipram on neutrophil CXCL1-induced shape change and CD11b expression. (A–D) Whole blood from healthy subjects (solid circles; n = 7), smokers (open circles; n = 5), and patients with COPD (solid triangles; n = 5) was preincubated with either 1 μM formoterol for 10 minutes followed by roflumilast N-oxide (A and C) or rolipram (B and D) for 10 minutes before measurements of neutrophil shape change toward CXCL1 (30 nM) (A and B) or expression of CD11b (C and D) using flow cytometry. Data are presented as mean ± SEM as a percentage of the maximum stimulation in the absence of roflumilast N-oxide or rolipram.
[More] [Minimize]Having established the effect of PDE4 inhibitors on neutrophil migratory responses, it was important to understand whether this could be due to elevation of intracellular cAMP. Therefore, because the responses to agonists and PDE4 inhibitors were similar in all cells examined, neutrophils from healthy subjects were incubated with the stable cAMP analog 8-Br-cAMP, the selective Epac activator 8-pCPT-2′-O-Me-cAMP, or the PKA-activator 6-BnZ-cAMP, and migration and [Ca2+]i responses to CXCL1 and LTB4 were measured. Both 8-Br-cAMP and 8-pCPT-2′-O-Me-cAMP inhibited migration to both CXCL1 and LTB4 completely in a concentration-dependent manner (Figures 6A and 6B and Table 2). However, both compounds failed to inhibit the [Ca2+]i response to CXCL1 (Figure 6C) but significantly inhibited the LTB4 response at 1 μM (8-Br-cAMP) and 10 μM 8-pCPT-29-O-Me-cAMP by 40–50%, respectively (Figure 6D). In contrast, the PKA activator failed to inhibit the migratory responses of neutrophils to either agonist (Figures 6A and 6B) and appeared to enhance the migration toward CXCL1 (Figure 6A); in addition, there was no effect on the [Ca2+]i responses. To confirm that this effect was mediated via Epac1 and not Epac2, the amount of expression of these isoforms was compared in neutrophils. The amount of Epac2 was almost undetectable by quantitative reverse transcriptase PCR, but Epac1 could be detected. However, there were no differences in expression between the subject groups (Figure E1). In an attempt to discriminate whether 8-pCPT-2′-O-Me-cAMP was acting via Epac1 or Epac2, selective Epac inhibitors were used to investigate whether the inhibitory effect of roflumilast could be reversed. The pan-Epac inhibitor ESI-09 reversed roflumilast-mediated inhibition of CXCL1-induced neutrophil migration, but the selective Epac2 inhibitor HJC0350 did not (Figure 6E). In contrast, the selective Epac1 inhibitor CE3F4 also reversed roflumilast-mediated inhibition of chemotaxis, suggesting that Epac1 may mediate this activity.
Figure 6. Effect of 8-bromoadenosine 3′,5′-cAMP (8-Br-cAMP), N6-benzoyladenosine-3′,5′-cAMP (6-Bnz-cAMP), or 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cAMP (8-pCPT-2′-O-Me-cAMP) on neutrophil migration and [Ca2+]i toward CXCL1 and LTB4. Neutrophils were isolated from the blood of control subjects and preincubated with either 8-Br-cAMP (solid circles), 8-pCPT-2′-O-Me-cAMP (open circles), or 6-Bnz-cAMP (solid triangles) for 1 hour before measurement of chemotaxis toward (A) CXCL1 (3 nM) or (B) LTB4 (1.3 nM) or of [Ca2+]i toward (C) CXCL1 (10 nM) or (D) LTB4 (30 nM). Data are presented as mean ± SEM as a percentage of the maximum chemotactic or peak calcium response in the absence of drug (n = 4–9). *P < 0.05 vs. control. (E) Neutrophils migrated toward CXCL1 in the absence or presence of roflumilast (1 nM) and either ESI-09 (10 μM), CE3F4 (20 μM), or HJC0350 (20 μM) for 1 hour. Data are presented as mean ± SEM (n = 4). ***P < 0.001 for differences from CXCL1 using ANOVA followed by Dunnett’s posttest. HJC = HJC0350; NS = not stimulated; ROF = roflumilast.
[More] [Minimize]| CXCL1 | LTB4 | |||
|---|---|---|---|---|
| Chemotaxis | [Ca2+]i | Chemotaxis | [Ca2+]i | |
| 8-Br-cAMP | 4.8 ± 3.8 nM | NC | 3.0 ± 2.2 nM | 238 ± 157 nM |
| n = 5 | n = 4 | n = 7 | ||
| 8-pCPT-2′-O-Me-cAMP | 13.7 ± 6.6 nM | NC | 3.3 ± 2.0 nM | 16.3 ± 10.3 nM |
| n = 5 | n = 4 | n = 8 | ||
Increased neutrophilia is a hallmark of COPD and is believed to drive many of the pathological features of the disease, owing to increased release of proteases, including neutrophil elastase and other serine proteases. This study shows that neutrophils from patients with COPD exhibit increased migratory activity toward various chemotactic stimuli, confirming our earlier observation with respect to LTB4 (21), but we now demonstrate that this enhanced migratory response also occurs with the chemokine CXCL1. This is similar to the responses we have reported for peripheral blood mononuclear cells from patients with COPD to this agonist (29), suggesting that there may be a general alteration in the response of leukocytes to chemotactic stimuli in COPD. Aberrant neutrophil migration by cells from patients with COPD has been reported by others (20, 22), although this is associated with a lack of directional cell movement rather than an increased chemotactic response, but these cells have an increased velocity (22). These differences may reflect the types of assays used (Boyden chambers vs. video microscopy), but both types of assays suggest alterations in neutrophil behavior in COPD. Neutrophil function and migration also alter with age (32), and the patients with COPD in this study were older than subjects in the control groups; however, this is reported to impair migration and not to increase migration. Nevertheless, it is possible that COPD neutrophils are “primed” in the circulation and therefore respond differently to stimuli.
There is a lack of novel antiinflammatory therapies for the treatment of COPD, although roflumilast has shown some promise. However, there are side effects associated with administration, including nausea, diarrhea, headaches, and weight loss (9, 33, 34), that appear to be directly linked to PDE4 inhibition (35). Studies in patients with COPD have demonstrated inhibition of sputum neutrophilia (13) and shown that this effect is sustained in patients selected for chronic bronchitis (9). Furthermore, a recent study showed that roflumilast was of benefit in acute exacerbations of COPD and also reduced sputum neutrophilia (36). There have been several mechanisms proposed to explain how roflumilast may be mediating antiinflammatory activity in COPD, including inhibition of inflammatory mediator release from airway cells (14, 37, 38) or inhibition of prolyl endopeptidase activity and hence a reduction in the breakdown of extracellular matrix to the neutrophil chemotactic factor N-acetyl proline-glycine-proline (39).
The present study proposes a mechanism whereby roflumilast may be exerting some of these antiinflammatory effects by suppressing neutrophil chemotaxis directly. Previously, others have demonstrated that roflumilast can reverse the corticosteroid insensitivity observed in neutrophils from patients with COPD (40), indicating that these cells can be direct targets for PDE4 inhibitors. This is supported by other data indicating that PDE4 inhibition leads to suppression of degranulation and adhesion to endothelial cells (18). The present study directly addressed whether neutrophil migration can be suppressed by PDE4 inhibition. The effects of roflumilast on neutrophil migration have been reported previously (17), but not in the context of COPD. In the present study, we demonstrated that roflumilast suppressed neutrophil migration to both LTB4 and CXCL1 in cells from patients with COPD with potency and efficacy similar to that seen in cells from control subjects and that it was more potent than another PDE4 inhibitor, rolipram. Classically, chemotactic receptors signal via G protein–coupled receptors that lead to increased [Ca2+]i, and we showed clearly that PDE4 inhibition also suppressed this intracellular signaling pathway, although the concentration–response curve was shifted to the right, indicating that the relationship between migration and changes in [Ca2+]i was not necessarily direct. This was most apparent when the cellular response to LTB4 was examined and showed that in COPD neutrophils, both roflumilast and rolipram failed to suppress [Ca2+]i. Such diversity in the relationship between changes in migration and [Ca2+]i has implications for screening assays in which inhibition of [Ca2+]i is often used as a surrogate for chemotaxis. Similarly, the response of cells to both shape change and expression of CD11b was also shifted to the right compared with the effect of PDE4 inhibition on chemotaxis. However, these effects on shape change and upregulation of CD11b are compatible with data from other studies (15) and may reflect binding of chemokine or drugs to other proteins in the blood sample (e.g., Duffy antigen), although the shape change assays required the addition of formoterol to increase intracellular cAMP and may be a reflection of sensitivity of this assay. Taken together, these data suggested the possibility that the effect of PDE4 inhibition on neutrophil chemotaxis toward CXCL1 and LTB4 may be partially independent of any effect on [Ca2+]i, and therefore alternative mechanisms were investigated.
The suppression of migration by PDE4 inhibition appeared to be due to increased intracellular cAMP because 8-Br-cAMP also inhibited chemotaxis to both CXCL1 and LTB4. Because cAMP directly activates Epac1, it was possible that the effects of roflumilast were via activation of this protein. This was confirmed by the suppression of chemotaxis by the Epac activator 8-pCPT-2′-O-Me-cAMP and further substantiated by reversal of roflumilast-mediated inhibition of chemotaxis by the Epac inhibitor ESI-09 and the selective Epac1 inhibitor CE3F4 but not the selective Epac2 inhibitor HJC0350. The role of Epac1 in this response differs from that in a murine model of cigarette smoke–induced pulmonary inflammation, whereby inflammation was suppressed in the Epac2-knockout mice but not in the Epac1-knockout mice (41). The mechanism underlying this discrepancy is not known, but we have demonstrated little expression of Epac2 in human neutrophils, and as such, the data in the animal model may be due to reduced chemokine expression rather than to changes in leukocyte migration. Elevation of cAMP can also lead to activation of PKA, and in neutrophils, this leads to a reduction in phagocytosis. However, activation of Epac can overcome this effect (42), as well as suppressing oxidative burst (43), thereby suggesting that a direct activator of this protein could have multiple beneficial effects in COPD. These data were further supported by the observation that the direct PKA activator 6-Bnz-cAMP, which does not activate Epac1, did not elicit similar reduction in migratory responses in these cells.
This study offers a possible mechanism for the reduction of airway neutrophilia observed in patients with COPD who have received roflumilast: by directly attenuating recruitment of cells into the lungs. The inhibitory effect of roflumilast N-oxide is entirely consistent with plasma concentrations that are achieved by oral administration of roflumilast (44). The clinical effect of roflumilast in patients with COPD is consistent with oral administration, which may prevent recruitment of neutrophils from the circulation into the lungs, and this may also explain why inhaled PDE4 inhibitors have proved to be ineffective in patients with COPD (45) and have been unable to inhibit the inflammatory profile in sputum, including neutrophilia (46). It appears that for PDE4 inhibitors to act as antiinflammatory agents, these drugs must be systemic and act directly on neutrophils. We have shown that the inhibitory effect of PDE4 inhibitors on neutrophil function may occur via direct activation of Epac1, thereby suggesting a novel target for drug therapy that would be independent of PDE4 inhibition and the associated side effect profile, as well as the deleterious effect on neutrophil function associated with elevation of intracellular cAMP. Such a drug could have benefit in diseases such as COPD, where there is considerable neutrophilic inflammation that is currently untreated by common antiinflammatory therapies.
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* L.E.D. is Associate Editor of AJRCMB. Her participation complies with American Thoracic Society requirements for recusal from review and decisions for authored works.
Supported by Takeda GmbH and the National Institute for Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London.
Author Contributions: Conception and design: L.E.D. and P.J.B.; performed experiments: A.E.D., T.K., P.S.F., C.M.D., and H.T.; data analysis and interpretation: A.E.D., T.K., P.S.F., C.M.D., H.T., and L.E.D.; drafting of the manuscript for important intellectual content: L.E.D. and P.J.B.
This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Originally Published in Press as DOI: 10.1165/rcmb.2018-0065OC on November 5, 2018
Author disclosures are available with the text of this article at www.atsjournals.org.
