Inhibition of airway hyperresponsiveness and pulmonary inflammation by roflumilast and other PDE4 inhibitors

Roflumilast is an oral, once-daily phosphodiesterase 4 (PDE4) inhibitor with anti-inflammatory activity. We compared the anti-inflammatory effects of roflumilast with those of PDE4 inhibitors rolipram, piclamilast, and cilomilast in ovalbumin (OVA)-sensitized and challenged Brown-Norway rats. Animals were treated orally 1h before OVA challenge with roflumilast (0.3, 1.0, and 3.0mg/kg), rolipram (0.8, 2.8, and 8.3mg/kg), piclamilast (10.0, 20.0, and 30.0mg/kg), or cilomilast (10.3, 34.3, and 103.0mg/kg). Airway hyperresponsiveness (AHR) against adenosine was investigated by measuring airway resistance 200min after OVA challenge. Subsequently, neutrophil influx and tumor necrosis factor-alpha (TNF-alpha) release in the lungs were determined by bronchoalveolar lavage. Direct bronchodilation at the time point of AHR assessment by PDE4 inhibitors was examined in serotonin-challenged animals. Evaluation of neutropenic animals or treatment with anti-TNF-alpha antibody revealed that AHR was independent of neutrophil accumulation or TNF-alpha release. Roflumilast (50% inhibitory dose [ID(50)]=1.5mg/kg) inhibited AHR 3-, 16-, and 27-fold more potently than rolipram, piclamilast, and cilomilast, respectively. Likewise, roflumilast was a more potent inhibitor of neutrophil influx (ID(50)=0.9mg/kg) than rolipram (ID(50)=6.9mg/kg), piclamilast (ID(50)=28.1mg/kg), or cilomilast (ID(50)=37.7mg/kg). Roflumilast, rolipram, and piclamilast-but not cilomilast-suppressed OVA-induced TNF-alpha release in a dose-dependent manner. Roflumilast (ID(50)=0.9mg/kg) exhibited 9- and 23-fold more potent inhibition of TNF-alpha release than rolipram and piclamilast, respectively. Roflumilast did not inhibit serotonin-induced bronchoconstriction 4.5h after administration, suggesting that inhibition of AHR by roflumilast results from anti-inflammatory, not bronchodilatory, effects. This study suggests that roflumilast has anti-inflammatory action and provides rationale for the investigation of roflumilast in asthmatic patients.     

A biochemical and functional assessment of monocyte phosphodiesterase activity in healthy and asthmatic subjects

The aim of this study was to investigate whether cyclic adenosine 3'5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 +/- 7.0, n=9) compared to healthy individuals (46.3 +/- 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF alpha release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF alpha release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.     

Induction of matrix metalloproteinases-14 and -2 by cyclical mechanical stretch is mediated by tumor necrosis factor-alpha in cultured human umbilical vein endothelial cells

OBJECTIVE: Mechanical forces have profound effects on endothelial cells. This study was undertaken to examine the hypothesis that tumor necrosis factor-alpha (TNF-alpha) is a potential mediator of stretch-induced effects on matrix metalloproteinase (MMP). METHODS: Human umbilical vein endothelial cells (HUVECs) grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. We used the TNF-alpha monoclonal antibody and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, to investigate the cyclical stretch-induced expression of MMP-14 and -2 in cultured HUVECs. RESULTS: Cyclical mechanical stretch significantly increased protein synthesis and mRNA expression for MMP-14 and -2 from 2 to 24 h. The increased MMP-14 and-2 proteins after stretch were completely blocked after the addition of TNF-alpha monoclonal antibody (5 microg/ml) or SP600125 (20 microM) 30 min before stretch. By zymography, MMP-2 expression was induced by cyclical stretch and was attenuated by TNF-alpha monoclonal antibody and SP600125. Cyclical stretch increased the immunohistochemical labeling of MMP-14 and -2 and significantly increased release of TNF-alpha into the culture media from 120+/-2 to 331+/-2 pg/ml (P<0.001) after stretch for 12 h. Cyclical stretch increased and SP600125 decreased the phosphorylated JNK. Gel-shifting assay showed that DNA-protein binding activity of AP-1 increased after cyclical stretch and TNF-alpha monoclonal antibody and SP600125 abolished the binding activity induced by cyclical stretch. CONCLUSION: These findings indicate that cyclical stretch augments TNF-alpha production and MMP genes expression in HUVECs. TNF-alpha mediates the stretch-induced MMP genes expression, at least in part, through the JNK pathway.     

Transvascular and Interstitial Migration of Neutrophils in Rat Mesentery

Objective : The consequential implications of the leukocyte-endothelial cell adhesion reaction, as well as the subsequent migratory activity of the leukocytes within the interstitium proper, are currently known only in general phenomenological terms. Our objective was to carry out a detailed quantitative analysis of transendothelial and interstitial migration. Method : We incorporated several new features, such as the video recording of time-lapse photography in conjunction with the time history of individual leukocytes under the influence of representative stimulators and inhibitors. The mesentery was suffused with the chemical activator N-formyl-met-leu-phe (fMLP, 5 ± 10^?8 M) and histamine (10^?6 M) for periods up to 3 h. This leads to an attachment of neutrophils preferentially to the walls of small postcapillary venules followed by migration into the adjacent interstitium. Results : The time interval between the initial neutrophil adhesion to the luminal side of the venules and their first appearance on the abluminal side of the venule wall was about 25 min. The time interval for the actual physical migration across the endothelium was only about 1-2 min. There was an initial increase in the number of neutrophils migrating across the wall per unit time after the application of fMLP. However, this effect then gradually decreased over the course of 1 h. In the extravascular tissue, the neutrophils appeared to migrate along random pathways with an average trajectory away from the venular wall. When histamine (1 μM) was applied topically in conjunction with fMLP, the overall migratory flux across the venular wall increased, although the cells migrated comparatively short distances into the interstitium. After pretreatment with phalloidin (25 μg/kg), an actin cross-linking agent, neutrophil adhesion and migration in response to fMLP stimulation was reduced. The mast cell stimulator 48/80 (25 mg/ml) resulted in rapid degranulation, which was accompanied by an insignificant increase in neutrophil diapedesis. After fMLP stimulation there was reduced neutrophil emigration in the presence of mast cell inhibitors, such as lodoxamide (1 μg/kg) and ketotifin (1 μg/kg). Conclusions : Our results demonstrate that both the endothelial cells and interstitial mast cells are involved in the rate of transvascular migration of leukocytes across postcapillary venules and into the interstitium.     

粒细胞活化、组蛋白去乙酰化酶2与重症哮喘关系的临床研究

目的 了解粒细胞活化和组蛋白去乙酰化酶2 (histone deacetylase 2,HDAC2)表达与重症哮喘的关系,以探索早期预测和辨别重症哮喘的生物标志物.方法 采用ELISA法测定受试者血清粒细胞活化标志物髓过氧化物酶(myeloperoxidase,MPO)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)、中性粒细胞弹性蛋白酶(neutrophil elastase,NE)及嗜酸粒细胞阳离子蛋白(eosinophil cationicprotein,ECP)水平,Western-blot测定外周血单个核细胞(peripheral blood mononuclear cells,PBMCs) HDAC2活性,分别比较重症哮喘组、轻中度哮喘组及健康对照组各指标并进行统计学分析.结果 ①重症哮喘组受试者粒细胞活化指标MPO[(102.43±28.19)μg/L]、MMP-9[(992.97±176.44) ng/L]、NE[(9.40±0.99)μg/L]及ECP[(232.85±48.37) μg/L]浓度值均显著高于轻中度哮喘组[MPO、MMP-9、NE及ECP分别为:(48.56±8.78) μg/L、(554.35±99.03) ng/L、(8.01±1.27) μg/L、(105.30±26.93) μg/L,P值均＜0.05]和正常对照组[MPO、MMP-9、NE及ECP分别为:(37.46±9.93) μg/L、(374.00±79.08) ng/L、(4.66±0.64) μg/L、(40.87±15.35) μg/L,P＜0.05];轻中度哮喘组受试者外周血MMP-9、NE及ECP浓度值显著高于正常对照组(P＜0.05);轻中度哮喘组与正常对照组比较MPO浓度值差异无统计学意义(P＞0.05);②重症哮喘组PBMCs HDAC2活性(HDAC2/GAPDH:0.19±0.09)较轻中度哮喘组(0.60±0.08)及正常对照组(1.09±0.26)均显著降低,差异有统计学意义(P＜0.05);轻中度哮喘组PBMCs HDAC2活性较正常对照组低,差异有统计学意义(P＜0.05);③MPO、MMP-9、NE及ECP与FEV1％ pred之间呈负相关,双侧Pearson检验差异有统计学意义(r值分别为:-0.60、-0.75、-0.43和-0.67,P＜0.01);与FEV1/FVC之间呈负相关,双侧Pearson检验差异有统计学意义(r值分别为:-0.52、-0.60、-0.42和-0.50,P＜0.05);与ACT之间呈负相关,双侧Pearson检验差异有统计学意义(r值分别为:-0.69、-0.67、-0.50和-0.70,P＜0.01);④HDAC2与FEV1％pred及FEV1/FVC之间呈正相关,双侧Pearson检验差异有统计学意义(r值分别为0.68和0.61,P＜0.01);与ACT之间呈正相关,双侧Pearson检验差异有统计学意义(r=0.68,P＜0.01).结论 粒细胞活化指标MPO、MMP-9、NE和ECP升高提示重症哮喘患者存在中性粒细胞和嗜酸粒细胞活化,其程度显著高于轻中度哮喘患者;PBMCs HDAC2在重症哮喘组明显降低提示该类患者的皮质激素敏感性降低,激素治疗的效果可能不佳.     

Rac2 is critical for neutrophil primary granule exocytosis

Neutrophil degranulation is important in many inflammatory disorders, although the intracellular mechanisms underlying this process remain poorly understood. The Rho GTPase, Rac2, has been implicated in control of degranulation in earlier studies. We hypothesized that Rac2 selectively regulates neutrophil primary granule release. Using bone marrow and peritoneal exudate neutrophils from rac2(-/-) mice in comparison with similar cells from wild-type C57Bl/6 mice, we found that primary granule myeloperoxidase and elastase release was absent in Rac2(-/-) neutrophils in response to chemoattractant stimulation, cytochalasin B/f-Met-Leu-Phe (CB/fMLP), and CB/leukotriene B4. Rac2(-/-) neutrophils also failed to exhibit mobilization of the primary granule marker CD63+ during CB/fMLP stimulation as determined by confocal microscopy. Priming of Rac2(-/-) neutrophils with tumor necrosis factor (TNF) or by peritoneal elicitation did not rescue the defect in primary granule release. However, phosphorylation of p38 mitogen-activated protein (MAP) kinase in Rac2(-/-) neutrophils was evident in response to CB/fMLP and/or TNF. Primary granule density and morphology were normal in Rac2(-/-) neutrophils. Secondary specific and tertiary granule release, measured by lactoferrin immunoassay and zymography, was normal in response to CB/fMLP and adhesion to fibronectin. These findings suggest an obligatory role for Rac2 in regulation of primary granule release by neutrophils.     

The influence of autoantibodies to myeloperoxidase on neutrophil function and intracellular signaling

Autoantibodies to myeloperoxidase (MPO) are associated with small vessel systemic vasculitis. Interactions of these autoantibodies with MPO target antigen, Fcgamma receptors and Beta2 integrins at the neutrophil surface, can set in train a sequence of intracellular signal transduction events that culminate with functional responses. These include a respiratory burst with release of superoxide ions, degranulation, cytokine release, enhanced adhesion and induction of an accelerated apoptotic program.     

Metalloproteinase-9 deficiency protects against hepatic ischemia/reperfusion injury

Leukocyte transmigration across endothelial and extracellular matrix protein barriers is dependent on adhesion and focal matrix degradation events. In the present study we investigated the role of metalloproteinase-9 (MMP-9/gelatinase B) in liver ischemia/reperfusion (I/R) injury using MMP-9-deficient (MMP-9(-/-)) animals and mice treated with a specific anti-MMP-9 neutralizing antibody or with a broad gelatinase inhibitor for both MMP-9 and metalloproteinase-2 (MMP-2/gelatinase A). Compared to wild-type mice, MMP-9(-/-) mice and mice treated with an anti-MMP-9 antibody showed significantly reduced liver damage. In contrast, mice treated with a broad gelatinase inhibitor showed rather inferior protection against I/R injury and were characterized by persistent ongoing liver inflammation, suggesting that MMP-2 and MMP-9 may have distinct roles in this type of injury. MMP-9 was mostly detected in Ly-6G and macrophage antigen-1 leukocytes adherent to the vessel walls and infiltrating the damaged livers of wild-type mice after liver I/R injury. Leukocyte traffic and cytokine expression were markedly impaired in livers of MMP-9(-/-) animals and in livers of mice treated with anti-MMP-9 antibody after I/R injury; however, initiation of the endothelial adhesion cascades was similar in both MMP-9(-/-) and control livers. We also showed that MMP-9-specific inhibition disrupted neutrophil migration across fibronectin in transwell filters and depressed myeloperoxidase (MPO) activation in vitro. CONCLUSION: These results support critical functions for MMP-9 in leukocyte recruitment and activation leading to liver damage. Moreover, they provide the rationale for identifying inhibitors to specifically target MMP-9 in vivo as a potential therapeutic approach in liver I/R injury.     

Patterns of primary degranulation as indicated by the mean myeloperoxidase index (MPXI) during bacteraemia in lymphoma transplants treated with growth factors.

The pattern of changes in neutrophil myeloperoxidase (MPO) before, during and after bacteraemia was studied in 34 patients recovering from autologous bone marrow transplantation for relapsed Hodgkin's disease and non Hodgkin's lymphomas. Thirteen patients received haemopoietic growth factors (7 received M-CSF, 3 received G-CSF and 3 GM-CSF). The mean peroxidase index (MPXI) produced as part of a routine FBC performed by a flow cytochemistry blood autoanalyser (Technicon H*1) was used as a parameter to assess the MPO and subsequently the azurophil degranulation. The manufacturer's normal values for MPXI range from -10 to +10. Median MPXI on the day of documented bacteraemia was just below normal in the control and M-CSF groups (-10.8 and -8.9 respectively), but it was much below normal in the G-CSF (-16.5, P < 0.05) and even lower in the GM-CSF group (-39.6, P < 0.02); this correlated well with the decreased bacteraemia incidence in the last two groups. Although contact of neutrophils with bacterial chemoattractants resulted in primary degranulation in all groups, the pattern of changes in MPO content was different, suggesting that neutrophils primed in vivo with various haemopoietins respond to the challenge of microbial agents via different pathways.     

Patterns of primary degranulation as indicated by the mean myeloperoxidase index (MPXI) during bacteraemia in lymphoma transplants treated with growth factors

Summary The pattern of changes in neutrophil myeloperoxidase (MPO) before, during and after bacteraemia was studied in 34 patients recovering from autologous bone marrow transplantation for relapsed Hodgkin's disease and non Hodgkin's lymphomas. Thirteen patients received haemopoietic growth factors (7 received M-CSF, 3 received G-CSF and 3 GM-CSF). The mean peroxidase index (MPXI) produced as part of a routine FBC performed by a flow cytochemistry blood autoanalyser (Technicon H*1) was used as a parameter to assess the MPO and subsequently the azurophil degranulation. The manufacturer's normal values for MPXI range from ?10 to +10. Median MPXI on the day of documented bacteraemia was just below normal in the control and M-CSF groups (?10.8 and ?8.9 respectively), but it was much below normal in the G-CSF (?16.5, P < 0.05) and even lower in the GM-CSF group (?39.6, P < 0.02); this correlated well with the decreased bacteraemia incidence in the last two groups. Although contact of neutrophils with bacterial chemoattractants resulted in primary degranulation in all groups, the pattern of changes in MPO content was different, suggesting that neutrophils primed in vivo with various haemopoietins respond to the challenge of microbial agents via different pathways.     

Constitutive mobilization of CD34+ cells into the peripheral blood in idiopathic myelofibrosis may be due to the action of a number of proteases

Idiopathic myelofibrosis (IM) is characterized by increased numbers of CD34(+) cells in the peripheral blood (PB). We explored the possible mechanisms underlying this abnormal trafficking of CD34(+) cells. Plasma levels of neutrophil elastase (NE), total and active matrix metalloproteinase 9 (MMP-9), and soluble vascular cell adhesion molecule-1 (sVCAM-1) were dramatically increased in IM. The absolute number of CD34(+) cells in the PB was correlated with the levels of sVCAM-1. Marked elevations of the levels of NE but not total and active MMP-9 as well as MMP-2 were detected in media conditioned by IM mononuclear cells (MNCs) as compared with that of healthy volunteers. IM MNC-conditioned media, however, was shown by zymographic analysis to contain increased gelatinolytic activity corresponding to the molecular weight of MMP-9. IM MNC-conditioned media also exhibited a greater ability to cleave VCAM-1 and c-kit in vitro, consistent with the biologic actions of NE. In addition, the increased ability of IM PB CD34(+) cells to migrate through a reconstituted basement membrane was diminished by several inhibitors of MMP-9 activity, indicating that these cells express increased levels of this MMP. These data indicate that a proteolytic environment exists in IM which might result in the sustained mobilization of CD34(+) cells.     

Effect of low-dose theophylline on airway inflammation in COPD

OBJECTIVE: Recent studies have shown that theophylline may exert anti-inflammatory effects on neutrophils. We undertook to assess the effect of theophylline on airway inflammation in COPD. METHODOLOGY: We performed a 4-week randomized double-blind, placebo-controlled study in 11 theophylline-naive patients with mild to moderate COPD. After a 1-week run-in period, six subjects were administered 400 mg/day theophylline (Theodur; Nikken Chemicals Co. Ltd, Tokyo, Japan) for 4 weeks, while five subjects were administered a placebo. Induced sputum was obtained before and after the run-in period and then after 2 and 4 weeks of treatment. Cell differential count and levels of interleukin-8, matrix metalloproteinase-9, neutrophil elastase (NE), myeloperoxidase (MPO), alpha1-antitrypsin (alpha1-AT), leukotriene B4 and tissue inhibitor of metalloproteinases-1 (TIMP-1) were assessed. RESULTS: No variable was significantly different during the run-in period or with placebo treatment. In contrast, theophylline treatment significantly decreased NE and MPO levels at 4 weeks, although the cell differential count did not change appreciably as a result of treatment. CONCLUSION: These results suggest that 4 weeks of theophylline treatment attenuates neutrophil-associated inflammation in the airways of mild to moderate COPD patients. However, the clinical benefits remain to be determined.     

Intercellular adhesion molecule-1 is required for chemoattractant-induced leukocyte adhesion in resting, but not inflamed, venules in vivo

The leukocyte integrins LFA-1 and Mac-1 bind to endothelial intercellular adhesion molecule-1 (ICAM-1). Leukocyte adhesion induced by micropipette injection of formylmethionylleucylphenylalanine (fMLP) or macrophage inflammatory protein 2 (MIP-2) next to a venule in the exteriorized mouse cremaster muscle was almost completely blocked after intravenous injection of the ICAM-1 mAb YN-1. In contrast, after 2-h pretreatment with TNF-alpha, leukocyte adhesion induced in postcapillary venules by fMLP or MIP-2 was not blocked by the ICAM-1 mAb. Leukocyte adhesion was significantly reduced by mAb GAME-46 to CD18 even after TNF-alpha treatment. We conclude that ICAM-1 is necessary for neutrophil adhesion to unstimulated endothelium, but not for adhesion to cytokine-stimulated endothelium. Although ICAM-1 is expressed at high levels after TNF-alpha, ICAM-1 either is not functional or is redundant with other endothelial ligands for beta(2) integrins.     

The novel phosphodiesterase 4 inhibitor, CI-1044, inhibits LPS-induced TNF-alpha production in whole blood from COPD patients

Chronic obstructive pulmonary disease (COPD) is a common, progressive respiratory disease that causes great morbidity and mortality despite treatment. Tumor necrosis factor alpha (TNF-alpha) plays a central role as a pro-inflammatory cytokine in COPD. TNF-alpha release is markedly inhibited by phosphodiesterase type 4 (PDE4) inhibitors that have proven efficacious in COPD clinical trials. The aim of this study was to compare the in vitro activities of the novel selective PDE4 inhibitors CI-1044 compared to well-known PDE4 inhibitors, rolipram and cilomilast, and to the glucocorticoid dexamethasone at reducing lipopolysaccharide (LPS)-induced TNF-alpha release in whole blood from COPD patients and healthy subjects. In the whole blood from COPD patients pre-incubation with PDE4 inhibitors or dexamethasone resulted in a dose-dependent inhibition of LPS-induced TNF-alpha release with IC(50) values of 1.3+/-0.7, 2.8+/-0.9 microM, higher to 10 microM and lesser than 0.03 microM for CI-1044, rolipram, cilomilast and dexamethasone, respectively. We observed a similar inhibition in the whole blood from healthy volunteers with, however, higher IC(50) values. These results indicate that CI-1044 inhibits in vitro LPS-induced TNF-alpha release in whole blood from COPD patients better than rolipram and cilomilast and suggested that it could be a useful anti-inflammatory therapy in COPD.     

Granulocyte markers in hypertonic and isotonic saline-induced sputum of asthmatic subjects.

The aim of this study was to assess whether hyperosmolarity affects granulocyte mediator levels in induced sputum of asthmatic subjects. A total of 32 mild-to-moderate asthmatics, who inhaled either hypertonic (HS; 4.5% NaCl) or isotonic (IS; 0.9% NaCl) solutions for 15 min, were studied. Selected sputum was used for analysis. Eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO) and free neutrophil elastase (NE) were measured in sputum supernatant. Sample weight, total and differential cell counts, as well as viability and squamous cell percentage were no different after the two tests. No significant differences in ECP, EPX, MPO or NE levels were observed between HS- and IS-induced sputum. Repeatability of the two tests was good for macrophages, neutrophils, eosinophils, ECP, EPX and NE, but not for lymphocytes and MPO. In conclusion, hyperosmolarity does not affect sputum cell counts and the levels of most granulocyte degranulation markers examined in this study, confirming that both hypertonic and isotonic solutions can be reliably used to induce sputum in asthmatics.     

Characterization of hematopoietic progenitor mobilization in protease-deficient mice

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE x CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.     

PDE4 inhibitors in COPD--a more selective approach to treatment

Chronic obstructive pulmonary disease (COPD) is a serious and mounting global public health problem. Although its pathogenesis is incompletely understood, chronic inflammation plays an important part and so new therapies with a novel anti-inflammatory mechanism of action may be of benefit in the treatment of COPD. Cilomilast and roflumilast are potent and selective phosphodiesterase (PDE)4 inhibitors, with an improved therapeutic index compared with the weak, non-selective PDE inhibitor, theophylline. Unlike theophylline, which is limited by poor efficacy and an unfavourable safety and tolerability profile, the selective PDE4 inhibitors are generally well tolerated, with demonstrated efficacy in improving lung function, decreasing the rate of exacerbations and improving quality of life, with proven anti-inflammatory effects in patients with COPD. Theophylline is a difficult drug to use clinically, requiring careful titration and routine plasma monitoring due to the risk of toxic side effects, such as cardiovascular and central nervous system adverse events, with dose adjustments required in many patients, including smokers, the elderly and some patients on concomitant medications. In contrast, the selective PDE4 inhibitors are convenient medications for both patient and physician alike. Hence these agents represent a therapeutic advance in the treatment of COPD, due to their novel mechanism of action and potent anti-inflammatory effects, coupled with a good safety and tolerability profile.     

Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease

Inhibitors of phosphodiesterase type 4 (PDE4) act by increasing intracellular concentrations of cyclic AMP, which has a broad range of anti-inflammatory effects on various key effector cells involved in asthma and chronic obstructive pulmonary disease (COPD). The therapeutic ratio for PDE4 inhibitors is thought to be determined by selectivity on receptor subtypes for relative effects on PDE4B (anti-inflammatory) and PDE4D (emesis). The two main orally active PDE4 inhibitors in the late phase III of clinical development are cilomilast and roflumilast; the latter (and its active metabolite N-oxide) is more selective and potent with a superior therapeutic ratio. Studies on cilomilast in COPD based on bronchial biopsy material have shown a broad range of anti-inflammatory activity, and the available evidence on clinical outcomes for up to 6 months with cilomilast 15 mg twice daily and roflumilast 500 mug once daily have shown variable but significant effects on exacerbations and quality of life, with small improvements in measures of pulmonary function. Roflumilast has a better safety and tolerability profile than cilomilast, with the main adverse effects being nausea, diarrhoea, and abdominal pain. Roflumilast also has activity in asthma as assessed by its attenuation of allergen and exercise challenges, and it shows clinical efficacy equivalent to that of beclomethasone dipropionate 400 mug daily. The emerging results of clinical trials on PDE4 inhibitors in asthma and COPD should be interpreted with cautious optimism since much of the evidence has been published only in abstract form to date. The next few years should resolve important issues about the potential role of these drugs as oral non-steroidal anti-inflammatory therapy for asthma and COPD and their place in management guidelines. Ultimately, clinicians will want to know whether PDE4 inhibitors are anything more than expensive "designer" theophylline, the archetypal non-selective phosphodiesterase inhibitor.     

Modulation of TNF and GM-CSF release from dispersed human nasal polyp cells and human whole blood by inhibitors of different PDE isoenzymes and glucocorticoids

The aim of this study was to investigate the role of the inhibitors of different PDE isoenzymes (PDE 1-5) on the production of two pro-inflammatory cytokines - tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Two in vitro models were used to compare the antiinflammatory properties of PDE inhibitors with that of glucocorticoids. The effect on TNF release from diluted human blood following lipopolysaccharide (LPS from Salmonella abortus equi) stimulation as well as the GM-CSF and TNF release from human nasal polyp cells following allergic stimulation were investigated. Both models proofed to be well suited for the characterisation of the antiinflammatory properties of new chemical entities.In diluted human blood and dispersed human nasal polyp cells the induced TNF release was most potently suppressed by selective PDE4 inhibitors. Amrinone and milrinone, selective PDE3 inhibitors, suppressed TNF secretion to a lesser extent. The effects of theophylline (unspecific PDE inhibitor), vinpocetine (PDE1 inhibitor), EHNA (PDE2 inhibitor) and the PDE5 inhibitors zaprinast and E 4021 were weak. In human blood, the tested glucocorticoids beclomethasone, dexamethasone and fluticasone inhibited the LPS induced TNF release potently in a concentration dependent manner, whereas in dispersed human nasal polyp cells, the effect of the glucocorticoids on allergically induced TNF release, with the exception of dexamethasone, was much less pronounced. Glucocorticoids were the most potent inhibitors of GM-CSF release and the effect correlates well with the affinity to the glucocorticoid receptor. The selective PDE 4 inhibitors, and to a certain extent the PDE3 inhibitors amrinone and milrinone, reduced the GM-CSF release in a concentration dependent manner. In all investigations selective PDE4 inhibitors reduced TNF release to a much higher degree (4-10 fold) than GM-CSF release.