Small airway fibroblasts from patients with chronic obstructive pulmonary disease exhibit cellular senescence.

Small airway disease (SAD) is a key early-stage pathology of chronic obstructive pulmonary disease (COPD). COPD is associated with cellular senescence whereby cells undergo growth arrest and express the senescence-associated secretory phenotype (SASP) leading to chronic inflammation and tissue remodeling. Parenchymal-derived fibroblasts have been shown to display senescent properties in COPD, however small airway fibroblasts (SAFs) have not been investigated. Therefore, this study investigated the role of these cells in COPD and their potential contribution to SAD. To investigate the senescent and fibrotic phenotype of SAF in COPD, SAFs were isolated from nonsmoker, smoker, and COPD lung resection tissue (n = 9-17 donors). Senescence and fibrotic marker expressions were determined using iCELLigence (proliferation), qPCR, Seahorse assay, and ELISAs. COPD SAFs were further enriched for senescent cells using FACSAria Fusion based on cell size and autofluorescence (10% largest/autofluorescent vs. 10% smallest/nonautofluorescent). The phenotype of the senescence-enriched population was investigated using RNA sequencing and pathway analysis. Markers of senescence were observed in COPD SAFs, including senescence-associated ß-galactosidase, SASP release, and reduced proliferation. Because the pathways driving this phenotype were unclear, we used cell sorting to enrich senescent COPD SAFs. This population displayed increased p21CIP1 and p16INK4a expression and mitochondrial dysfunction. RNA sequencing suggested these senescent cells express genes involved in oxidative stress response, fibrosis, and mitochondrial dysfunction pathways. These data suggest COPD SAFs are senescent and may be associated with fibrotic properties and mitochondrial dysfunction. Further understanding of cellular senescence in SAFs may lead to potential therapies to limit SAD progression.NEW & NOTEWORTHY Fibroblasts and senescence are thought to play key roles in the pathogenesis of small airway disease and COPD; however, the characteristics of small airway-derived fibroblasts are not well explored. In this study we isolate and enrich the senescent small airway-derived fibroblast (SAF) population from COPD lungs and explore the pathways driving this phenotype using bulk RNA-seq.

MicroRNA-570 is a novel regulator of cellular senescence and inflammaging.

Diseases of accelerated aging often occur together (multimorbidity), and their prevalence is increasing, with high societal and health care costs. Chronic obstructive pulmonary disease (COPD) is one such condition, in which one half of patients exhibit ≥4 age-related diseases. Diseases of accelerated aging share common molecular pathways, which lead to the detrimental accumulation of senescent cells. These senescent cells no longer divide but release multiple inflammatory proteins, known as the senescence-associated secretory phenotype, which may perpetuate and speed disease. Here, we show that inhibiting miR-570-3p, which is increased in COPD cells, reverses cellular senescence by restoring the antiaging molecule sirtuin-1. MiR-570-3p is induced by oxidative stress in airway epithelial cells through p38 MAP kinase-c-Jun signaling and drives senescence by inhibiting sirtuin-1. Inhibition of elevated miR-570-3p in COPD small airway epithelial cells, using an antagomir, restores sirtuin-1 and suppresses markers of cellular senescence (p16INK4a, p21Waf1, and p27Kip1), thereby restoring cellular growth by allowing progression through the cell cycle. MiR-570-3p inhibition also suppresses the senescence-associated secretory phenotype (matrix metalloproteinases-2/9, C-X-C motif chemokine ligand 8, IL-1ß, and IL-6). Collectively, these data suggest that inhibiting miR-570-3p rejuvenates cells via restoration of sirtuin-1, reducing many of the abnormalities associated with cellular senescence.-Baker, J. R., Vuppusetty, C., Colley, T., Hassibi, S., Fenwick, P. S., Donnelly, L. E., Ito, K., Barnes, P. J. MicroRNA-570 is a novel regulator of cellular senescence and inflammaging.

Human Rhinovirus Impairs the Innate Immune Response to Bacteria in Alveolar Macrophages in Chronic Obstructive Pulmonary Disease.

Rationale: Human rhinovirus (HRV) is a common cause of chronic obstructive pulmonary disease (COPD) exacerbations. Secondary bacterial infection is associated with more severe symptoms and delayed recovery. Alveolar macrophages clear bacteria from the lung and maintain lung homeostasis through cytokine secretion. These processes are defective in COPD. The effect of HRV on macrophage function is unknown. Objectives: To investigate the effect of HRV on phagocytosis and cytokine response to bacteria by alveolar macrophages and monocyte-derived macrophages (MDM) in COPD and healthy control subjects. Methods: Alveolar macrophages were obtained by bronchoscopy and MDM by adherence. Macrophages were exposed to HRV16 (multiplicity of infection 5), polyinosinic:polycytidylic acid (poly I:C) 30 µg/ml, IFN-ß 10 µg/ml, IFN-γ 10 µg/ml, or medium control for 24 hours. Phagocytosis of fluorescently labeled Haemophilus influenzae or Streptococcus pneumoniae was assessed by fluorimetry. CXCL8 (IL-8), IL-6, TNF-α (tumor necrosis factor-α), and IL-10 release was measured by ELISA. Measurements and Main Results: HRV significantly impaired phagocytosis of H. influenzae by 23% in MDM (n = 37; P = 0.004) and 18% in alveolar macrophages (n = 20; P < 0.0001) in COPD. HRV also significantly reduced phagocytosis of S. pneumoniae by 33% in COPD MDM (n = 20; P = 0.0192). There was no effect in healthy control subjects. Phagocytosis of H. influenzae was also impaired by poly I:C but not IFN-ß or IFN-γ in COPD MDM. HRV significantly reduced cytokine responses to H. influenzae. The IL-10 response to H. influenzae was significantly impaired by poly I:C, IFN-ß, and IFN-γ in COPD cells. Conclusions: HRV impairs phagocytosis of bacteria in COPD, which may lead to an outgrowth of bacteria. HRV also impairs cytokine responses to bacteria via the TLR3/IFN pathway, which may prevent resolution of inflammation leading to prolonged exacerbations in COPD.

Direct Inhibitory Effect of the PDE4 Inhibitor Roflumilast on Neutrophil Migration in Chronic Obstructive Pulmonary Disease.

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.

Enhanced monocyte migration to CXCR3 and CCR5 chemokines in COPD.

Chronic obstructive pulmonary disease (COPD) patients exhibit chronic inflammation, both in the lung parenchyma and the airways, which is characterised by an increased infiltration of macrophages and T-lymphocytes, particularly CD8+ cells. Both cell types can express chemokine (C-X-C motif) receptor (CXCR)3 and C-C chemokine receptor 5 and the relevant chemokines for these receptors are elevated in COPD. The aim of this study was to compare chemotactic responses of lymphocytes and monocytes of nonsmokers, smokers and COPD patients towards CXCR3 ligands and chemokine (C-C motif) ligand (CCL)5. Migration of peripheral blood mononuclear cells, monocytes and lymphocytes from nonsmokers, smokers and COPD patients toward CXCR3 chemokines and CCL5 was analysed using chemotaxis assays. There was increased migration of peripheral blood mononuclear cells from COPD patients towards all chemokines studied when compared with nonsmokers and smokers. Both lymphocytes and monocytes contributed to this enhanced response, which was not explained by increased receptor expression. However, isolated lymphocytes failed to migrate and isolated monocytes from COPD patients lost their enhanced migratory capacity. Both monocytes and lymphocytes cooperate to enhance migration towards CXCR3 chemokines and CCL5. This may contribute to increased numbers of macrophages and T-cells in the lungs of COPD patients, and inhibition of recruitment using selective antagonists might be a treatment to reduce the inflammatory response in COPD.

Identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease.

BACKGROUND: In patients with chronic obstructive pulmonary disease (COPD), pulmonary macrophages increase in number, release increased levels of inflammatory mediators, and respond poorly to glucocorticosteroids. Whether this is due to a change in macrophage phenotype or localized activation is unknown. OBJECTIVE: We sought to investigate whether macrophages from patients with COPD are a distinct phenotype. METHODS: Macrophage populations were isolated from human lung tissue from nonsmokers, smokers, and patients with COPD by using Percoll density gradients. Five macrophage populations were isolated on the basis of density (1.011-1.023, 1.023-1.036, 1.036-1.048, 1.048-1.061, and 1.061-1.073 g/mL), and cell-surface expression of CD14, CD16, CD163, CD40, and CD206 was assessed by using flow cytometry. Release of active matrix metalloproteinase 9, TNF-α, CXCL8, and IL-10 was measured by using ELISA. RESULTS: The 2 least dense fractions were more than 90% apoptotic/necrotic, with the remaining fractions greater than 70% viable. Macrophages from nonsmokers and smokers were CD163(+), CD206(+), CD14(+), and CD40(-), whereas macrophages from patients with COPD were less defined, showing significantly lower expression of all receptors. There were no differences in receptor expression associated with density. Macrophages from patients with COPD of a density of 1.036 to 1.048 g/mL released higher levels of active matrix metalloproteinase 9 compared with cells from nonsmokers, with no difference between the remaining fractions. This population of macrophages from patients with COPD was less responsive to budesonide compared with those from nonsmokers and smokers when stimulated with LPS. Glucocorticosteroid insensitivity was selective for proinflammatory cytokines because budesonide inhibition of LPS-stimulated IL-10 release was similar for all macrophages. CONCLUSIONS: This study identifies a specific macrophage phenotype in the lungs of patients with COPD who are glucocorticosteroid insensitive with a density of 1.036 to 1.048 g/mL but do not correspond to the current concept of macrophage phenotypes.

IL-36 receptor agonist and antagonist imbalance drives neutrophilic inflammation in COPD.

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared with control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and is further induced by a viral mimetic, whereas IL-36Ra is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ, thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, which was inhibited by exogenous IL-36Ra. The use of a therapeutic antibody that inhibits binding to the IL-36R attenuated IL-36γ-driven inflammation and cellular crosstalk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and have shown that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising therapeutic strategy in the treatment of COPD.

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress.

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H(2)O(2)) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H(2)O(2)-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r=0.92, p<0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.

Isorhapontigenin, a bioavailable dietary polyphenol, suppresses airway epithelial cell inflammation through a corticosteroid-independent mechanism.

BACKGROUND AND PURPOSE: Chronic obstructive pulmonary disease (COPD) is a corticosteroid-resistant airway inflammatory condition. Resveratrol exhibits anti-inflammatory activities in COPD but has weak potency and poor pharmacokinetics. This study aimed to evaluate the potential of isorhapontigenin, another dietary polyphenol, as a novel anti-inflammatory agent for COPD by examining its effects in vitro and pharmacokinetics in vivo. EXPERIMENTAL APPROACH: Primary human airway epithelial cells derived from healthy and COPD subjects, and A549 epithelial cells were incubated with isorhapontigenin or resveratrol and stimulated with IL-1ß in the presence or absence of cigarette smoke extract. Effects of isorhapontigenin and resveratrol on the release of IL-6 and chemokine (C-X-C motif) ligand 8 (CXCL8), and the activation of NF-κB, activator protein-1 (AP-1), MAPKs and PI3K/Akt/FoxO3A pathways were determined and compared with those of dexamethasone. The pharmacokinetic profiles of isorhapontigenin, after i.v. or oral administration, were assessed in Sprague-Dawley rats. KEY RESULTS: Isorhapontigenin concentration-dependently inhibited IL-6 and CXCL8 release, with IC50 values at least twofold lower than those of resveratrol. These were associated with reduced activation of NF-κB and AP-1 and, notably, the PI3K/Akt/FoxO3A pathway, that was relatively insensitive to dexamethasone. In vivo, isorhapontigenin was rapidly absorbed with abundant plasma levels after oral dosing. Its oral bioavailability was approximately 50% higher than resveratrol. CONCLUSIONS AND IMPLICATIONS: Isorhapontigenin, an orally bioavailable dietary polyphenol, displayed superior anti-inflammatory effects compared with resveratrol. Furthermore, it suppressed the PI3K/Akt pathway that is insensitive to corticosteroids. These favourable efficacy and pharmacokinetic properties support its further development as a novel anti-inflammatory agent for COPD.

Effect of JAK Inhibitors on Release of CXCL9, CXCL10 and CXCL11 from Human Airway Epithelial Cells.

BACKGROUND: CD8+ T-cells are located in the small airways of COPD patients and may contribute to pathophysiology. CD8+ cells express the chemokine receptor, CXCR3 that binds CXCL9, CXCL10 and CXCL11, which are elevated in the airways of COPD patients. These chemokines are released from airway epithelial cells via activation of receptor associated Janus kinases (JAK). This study compared the efficacy of two structurally dissimilar pan-JAK inhibitors, PF956980 and PF1367550, and the glucocorticosteroid dexamethasone, in BEAS-2B and human primary airway epithelial cells from COPD patients and control subjects. METHODS: Cells were stimulated with either IFNγ alone or with TNFα, and release of CXCL9, CXCL10 and CXCL11 measured by ELISA and expression of CXCL9, CXCL10 and CXCL11 by qPCR. Activation of JAK signalling was assessed by STAT1 phosphorylation and DNA binding. RESULTS: There were no differences in the levels of release of CXCL9, CXCL10 and CXCL11 from primary airway epithelial cells from any of the subjects or following stimulation with either IFNγ alone or with TNFα. Dexamethasone did not inhibit CXCR3 chemokine release from stimulated BEAS-2B or primary airway epithelial cells. However, both JAK inhibitors suppressed this response with PF1367550 being ~50-65-fold more potent than PF956980. The response of cells from COPD patients did not differ from controls with similar responses regardless of whether inhibitors were added prophylactically or concomitant with stimuli. These effects were mediated by JAK inhibition as both compounds suppressed STAT1 phosphorylation and DNA-binding of STAT1 and gene transcription. CONCLUSIONS: These data suggest that the novel JAK inhibitor, PF1367550, is more potent than PF956980 and that JAK pathway inhibition in airway epithelium could provide an alternative anti-inflammatory approach for glucocorticosteroid-resistant diseases including COPD.

Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes.

Early reports indicate that long non-coding RNAs (lncRNAs) are novel regulators of biological responses. However, their role in the human innate immune response, which provides the initial defence against infection, is largely unexplored. To address this issue, here we characterize the long non-coding RNA transcriptome in primary human monocytes using RNA sequencing. We identify 76 enhancer RNAs (eRNAs), 40 canonical lncRNAs, 65 antisense lncRNAs and 35 regions of bidirectional transcription (RBT) that are differentially expressed in response to bacterial lipopolysaccharide (LPS). Crucially, we demonstrate that knockdown of nuclear-localized, NF-κB-regulated, eRNAs (IL1ß-eRNA) and RBT (IL1ß-RBT46) surrounding the IL1ß locus, attenuates LPS-induced messenger RNA transcription and release of the proinflammatory mediators, IL1ß and CXCL8. We predict that lncRNAs can be important regulators of the human innate immune response.

The role of IkappaB kinase 2, but not activation of NF-kappaB, in the release of CXCR3 ligands from IFN-gamma-stimulated human bronchial epithelial cells.

The severity of chronic obstructive pulmonary disease correlates with increased numbers of cytotoxic CD8(+) T lymphocytes in the lung parenchyma. CD8(+) T lymphocytes release IFN-gamma which stimulates airway epithelial cells to produce CXCR3 chemokines leading to further recruitment of CD8(+) T lymphocytes. To evaluate the signaling pathways involved in regulation of CXCR3 ligands, the human bronchial epithelial cell line BEAS-2B was stimulated with IFN-gamma and the release of the CXCR3 ligands was measured by ELISA. The release of CXCL9, CXCL10, and CXCL11 was inhibited by an IkappaB kinase 2 (IKK2) selective inhibitor 2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) (EC(50) values were 0.50 +/- 0.03, 0.17 +/- 0.06, and 0.45 +/- 0.10 microM, respectively (n = 6)) and an IKK1/2 selective inhibitor 2-amino-6-(2'cyclopropylemethoxy-6'-hydroxy-phenyl)-4-piperidin-3-yl-pyridine-3-carbonitrile (EC(50) values 0.74 +/- 0.40, 0.27 +/- 0.06, and 0.88 +/- 0.29 microM, respectively (n = 6)). The glucocorticosteroid dexamethasone had no effect on CXCR3 ligand release. The release of CXCL10 was most sensitive to inhibition by IKK2 and a role for IKK2 in CXCL10 release was confirmed by overexpression of dominant-negative adenoviral constructs to IKK2 (68.2 +/- 8.3% n = 5), but not of IKK1. Neither phosphorylation of IkappaBalpha, translocation of p65 to the nucleus, or activation of a NF-kappaB-dependent reporter (Ad-NF-kappaB-luc) were detected following stimulation of BEAS-2B cells with IFN-gamma. These data suggest that IKK2 is also involved in the IFN-gamma-stimulated release of the CXCR3 ligands through a novel mechanism that is independent NF-kappaB.

Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells.

Peroxynitrite, formed by the reaction of nitric oxide (NO. ) with superoxide anions (O(2)(-).), may play a role in the pathophysiology of inflammation. The effects of 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, on the human bronchial epithelial cell line BEAS-2B, were examined. SIN-1 exposure resulted in cell death in a time- and dose-dependent manner. Depletion of intracellular glutathione increased the vulnerability of the cells. Pretreatment with Mn(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (MnTMPyP) or hydroxocobalamin (HC), O(2)(-). and NO. scavengers, respectively, reduced significantly SIN-1-induced cell death (18.66 +/- 3.57 vs. 77.01 +/- 14.07 or 82.20 +/- 9.64, % cell viability SIN-1 vs. MnTMPyP or HC). Moreover, the mitogen-activated protein kinases (MAPK) p44/42 (ERK), p38, and p54/46 (JNK) were also activated in a time- and concentration-dependent manner. PD-98059 and SB-239063, specific inhibitors of ERK and p38 MAPK pathways, failed to protect cells against 1 mM SIN-1. However, PD-98059 partially inhibited (60% cell survival) SIN-1 effects at < or =0.25 mM, and this was increased with the inclusion of SB-239063. Therefore, MAPKs may mediate signal transduction pathways induced by peroxynitrite in lung epithelial cells leading to cell death.

Anti-inflammatory effects of resveratrol in lung epithelial cells: molecular mechanisms.

Resveratrol (3,4',5-trihydroxystilbene) is a polyphenolic stilbene found in the skins of red fruits, including grapes, that may be responsible for some of the health benefits ascribed to consumption of red wine. Resveratrol has been shown to have antioxidant properties and can act as an estrogen agonist. This study examined the anti-inflammatory effects of resveratrol on human airway epithelial cells. Resveratrol and the related molecule quercetin, but not deoxyrhapontin, inhibited IL-8 and granulocyte-macrophage colony-stimulating factor release from A549 cells. Neither the estrogen receptor antagonist tamoxifen nor the glucocorticoid antagonist mifepristone altered the inhibitory effect of resveratrol. The mechanism of resveratrol action was investigated further using luciferase reporter genes stably transfected into A549 cells. Resveratrol and quercetin inhibited NF-kappaB-, activator protein-1-, and cAMP response element binding protein-dependent transcription to a greater extent than the glucocorticosteroid dexamethasone. These compounds also had no significant effect on acetylation or deacetylation of core histones. Resveratrol, but not estradiol or N-acetyl cysteine, inhibited cytokine-stimulated inducible nitric oxide synthase expression and nitrite production (IC50 = 3.6 +/- 2.9 microM) in human primary airway epithelial cells. Resveratrol also inhibited granulocyte-macrophage colony-stimulating factor release (IC50 = 0.44 +/- 0.17 microM), IL-8 release (IC50 = 4.7 +/- 3.3 microM), and cyclooxygenase-2 expression in these cells. This study demonstrates that resveratrol and quercetin have novel nonsteroidal anti-inflammatory activity that may have applications for the treatment of inflammatory diseases.

Discovery of BRL 50481 [3-(N,N-dimethylsulfonamido)-4-methyl-nitrobenzene], a selective inhibitor of phosphodiesterase 7: in vitro studies in human monocytes, lung macrophages, and CD8+ T-lymphocytes.

The biochemical and pharmacological characteristics in human proinflammatory cells of BRL 50481 [3-(N,N-dimethylsulfonamido)-4-methyl-nitrobenzene], a novel and selective inhibitor of phosphodiesterase (PDE) 7, are described. BRL 50481 inhibited the activity of hrPDE7A1 expressed in baculovirus-infected Spodoptera frugiperda 9 cells in a competitive manner (Ki value of 180 nM) and was 416 and 1884 times less potent against PDE3 and 38 and 238 times less potent against PDE4 at a substrate concentration of 1 microM and 50 nM cAMP, respectively. Western blotting identified HSPDE7A1 but not HSPDE7A2 in three human cell types that are implicated in the pathogenesis of chronic obstructive lung disease, namely, CD8+ T-lymphocytes, monocytes, and lung macrophages. BRL 50481 had no effect on the proliferation of CD8+ T-lymphocytes and only marginally (approximately 2-11%) reduced the generation of tumor necrosis factor (TNF)alpha from blood monocytes and lung macrophages. However, in the presence of BRL 50481 the inhibitory effect of rolipram was enhanced on all three cell types. The expression of HSPDE7A1 was increased in a time-dependent manner in monocytes that were "aged" in culture medium. Under this condition, BRL 50481 now inhibited TNFalpha generation in a concentration-dependent manner. In aged monocytes, rolipram, Org 9935 (a PDE3 inhibitor), and prostaglandin E2 inhibited TNFalpha generation in a concentration-dependent manner and interacted additively with BRL 50481. BRL 50481 is the first fully documented PDE7 inhibitor that has acceptable selectivity for in vitro studies. Furthermore, although BRL 50481 had only a modest inhibitory effect per se on the proinflammatory cells studied, it acted at least additively with other cAMP-elevating drugs, especially when HSPDE7A1 was up-regulated.