Lower airway bacterial colonisation (LABC) in COPD patients is associated with increased exacerbation frequency and faster lung function decline. Defective macrophage phagocytosis in COPD drives ...inflammation, but how defective macrophage function contributes to exacerbations is not clear. This study investigated the association between macrophage phagocytosis and exacerbation frequency, LABC and clinical parameters.
Monocyte-derived macrophages (MDM) were generated from 92 stable COPD patients, and at the onset of exacerbation in 39 patients. Macrophages were exposed to fluorescently labelled Haemophilus influenzae or Streptococcus pneumoniae for 4 h, then phagocytosis measured by fluorimetry and cytokine release by ELISA. Sputum bacterial colonisation was measured by PCR.
Phagocytosis of H. influenzae was negatively correlated with exacerbation frequency (r = 0.440, p < 0.01), and was significantly reduced in frequent vs. infrequent exacerbators (1.9 × 10
RFU vs. 2.5 × 10
RFU, p < 0.01). There was no correlation for S. pneumoniae. There was no association between phagocytosis of either bacteria with age, lung function, smoking history or treatment with inhaled corticosteroids, or long-acting bronchodilators. Phagocytosis was not altered during an exacerbation, or in the 2 weeks post-exacerbation. In response to phagocytosis, MDM from exacerbating patients showed increased release of CXCL-8 (p < 0.001) and TNFα (p < 0.01) compared to stable state.
Impaired COPD macrophage phagocytosis of H. influenzae, but not S. pneumoniae is associated with exacerbation frequency, resulting in pro-inflammatory macrophages that may contribute to disease progression. Targeting these frequent exacerbators with drugs that improve macrophage phagocytosis may prove beneficial.
BackgroundCOPD is associated with cellular senescence and fibrosis. Extracellular vesicles (EVs) are membrane-derived vesicles involved in intercellular communication. EVs contain miRNAs, mRNA and ...proteins and have been implicated in COPD to induce senescence and the transition of fibroblast to myofibroblasts. This study examined whether EVs derived from COPD fibroblasts drive senescence in healthy recipient fibroblasts. Changes in expression of p21CIP1 and alpha-smooth muscle actin (αSMA) were chosen as markers of senescence and transition of fibroblasts to myofibroblasts respectively.MethodsLarge EVs, and small EVs were isolated from media from non-smoker (NS) and COPD fibroblasts cultured with or without H2O2. EVs were labelled with phk67 and uptake measured by flow cytometry. Healthy recipient fibroblasts were cultured with EVs or EV-free media for 24h and 48h and protein expression of p21CIP1 and αSMA measured using western blots and CXCL8 release by ELISA.ResultsThere was a time-dependent uptake of EVs into recipient cells with no difference between EVs from control or COPD fibroblasts with 91.8 ± 3.8% of recipient cells phk67 positive by 48h (n=4). Incubation of recipient fibroblasts (n=2–5) with large EVs from either non-smokers or COPD subjects did not alter the expression of p21CIP1 or αSMA at 24h. Similarly large EVs from fibroblasts exposed to H2O2 had no effect on these markers in recipient cells. By contrast, at 48h (figure 1), small EVs from COPD cells showed a trend to increased expression of p21CIP1 and EVs from both non-smokers and COPD subjects increased expression of αSMA. Incubation of recipient cells with large EVs from non-smoker fibroblasts that had been cultured with or without H2O2 increased release of CXCL8 (0.36±0.15ng/ml to 5.43±3.92ng/ml and 5.44±5.23ng/ml respectively) and small EVs from COPD fibroblasts induced CXCL8 release at 48h (0.36±0.15ng/ml to 3.75±3.16ng/ml).ConclusionsLarge and small EVs tend to increase the expression of p21CIP1 and αSMA in recipient fibroblasts. These results are confirmed by the uptake analysis showing that maximum uptake of EVs from both NS and COPD fibroblasts is reached after 48h. Altogether, these data suggest that EVs participate in COPD pathophysiology by spreading senescence in recipient fibroblasts.Abstract P208 Figure 1Effect of Large and Small EVs on p21CIP1 and αSMA Expression in NS Fibroblasts Stimulated for 48h. Healthy fibroblasts from non-smoker (NS) subjects were incubated with large and small EVs derived from healthy NS or COPD fibroblasts, derived from cells that had been cultured in the absence or presence of 100µM H2O2. Cells were also stimulated with media only (NT) and media containing H2O2 as controls. Cells were lysed after 48h (a, d) and the expression of p21CIP1 (b, e) and αSMA (c, f) was measured relative to β-actin expression and data presented as mean±SEM. Representative blots are presented in panels a and d.
Background: The pathophysiology of chronic obstructive pulmonary disease (COPD) features pulmonary inflammation with a predominant alveolar macrophage involvement. Bronchoalveolar macrophages from ...patients with COPD release increased amounts of inflammatory cytokines in vitro, an effect that is not inhibited by the glucocorticosteroid dexamethasone. Resveratrol (3,5,4′-trihydroxystilbene) is a component of red wine extract that has anti-inflammatory and antioxidant properties. A study was undertaken to determine whether or not resveratrol would inhibit cytokine release in vitro by alveolar macrophages from patients with COPD. Methods: Alveolar macrophages were isolated from bronchoalveolar lavage (BAL) fluid from cigarette smokers and from patients with COPD (n=15 per group). The macrophages were stimulated with either interleukin (IL)-1β or cigarette smoke media (CSM) to release IL-8 and granulocyte macrophage-colony stimulating factor (GM-CSF). The effect of resveratrol was examined on both basal and stimulated cytokine release. Results: Resveratrol inhibited basal release of IL-8 in smokers and patients with COPD by 94% and 88% respectively, and inhibited GM-CSF release by 79% and 76% respectively. Resveratrol also inhibited stimulated cytokine release. Resveratrol reduced IL-1β stimulated IL-8 and GM-CSF release in both smokers and COPD patients to below basal levels. In addition, resveratrol inhibited CSM stimulated IL-8 release by 61% and 51% respectively in smokers and COPD patients, and inhibited GM-CSF release by 49% for both subject groups. Conclusions: Resveratrol inhibits inflammatory cytokine release from alveolar macrophages in COPD. Resveratrol or similar compounds may be effective pharmacotherapy for macrophage pathophysiology in COPD.
Pulmonary macrophages are a target for inhaled therapies. Combinations of long-acting beta(2)-agonists (LABA) and glucocorticosteroids have been developed for asthma and chronic obstructive pulmonary ...disease (COPD). This study examined two LABA, salmeterol and formoterol, and the glucocorticosteroid, budesonide, on cytokine release from monocyte-derived macrophages (MDM) to determine whether anti-inflammatory effects observed in patients are due to inhibition of macrophages. MDM were incubated in the absence or presence of LABA or budesonide prior to stimulation with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-alpha, granulocyte macrophage-colony stimulating factor (GM-CSF) and CXC chemokine ligand (CXCL)8 were measured by ELISA. Formoterol and salmeterol inhibited LPS-stimulated release of TNF-alpha (mean effective concentration (EC(50)) 2.4+/-1.8 and 3.5+/-2.7 nM, respectively; n = 11-16), GM-CSF (EC(50) 24.6+/-2.1 and 52.4+/-40.8 nM, respectively, n = 11-12) but not CXCL8 from LPS-stimulated MDM. Budesonide inhibited release of all three cytokines (EC(50) TNF-alpha: 1.2+/-0.4 nM; GM-CSF: 0.4+/-0.2 nM; CXCL8: 0.4+/-0.1 nM; n = 3-4). Formoterol but not salmeterol elevated cAMP in these cells. These effects were attenuated by beta-adrenoceptor antagonists, propranolol and ICI118551. Salmeterol (10(-7) M) also inhibited formoterol-induced cAMP and formoterol-mediated attenuation of cytokine release. Combining budesonide (0.3 nM) with formoterol, inhibited TNF-alpha release additively. LABA may inhibit inflammatory cytokine release from macrophages in a cAMP-independent manner and act additively with budesonide.
Macrophages increase in number and are highly activated in chronic obstructive pulmonary disease (COPD). Muscarinic receptor antagonists inhibit acetylcholine-stimulated release of neutrophilic ...chemoattractants, suggesting that acetylcholine may regulate macrophage responses. Therefore, expression and function of components of the non-neuronal cholinergic system in monocyte-macrophage cells was investigated. RNA was isolated from monocytes, monocyte-derived macrophages (MDMs), lung and alveolar macrophages from nonsmokers, smokers and COPD patients, and expression of the high-affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter and muscarinic receptors (M(1)-M(5)) ascertained using real-time PCR. M(2) and M(3) receptor expression was confirmed using immunocytochemistry. Release of interleukin (IL)-8, IL-6 and leukotriene (LT)B(4) were measured by ELISA or EIA. All monocyte-macrophage cells expressed mRNA for components of the non-neuronal cholinergic system. Lung macrophages expressed significantly more M(1) mRNA compared with monocytes, and both lung macrophages and alveolar macrophages expressed the highest levels of M(3) mRNA. Expression of M(2) and M(3) protein was confirmed in MDMs and lung macrophages. Carbachol stimulated release of LTB(4) from lung macrophages (buffer 222.3 ± 75.1 versus carbachol 1,118 ± 622.4 pg · mL(-1); n = 15, p<0.05) but not IL-6 or IL-8. LTB(4) release was attenuated by the M(3) antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; half maximal effective concentration 5.2 ± 2.2 nM; n = 9). Stimulation of macrophage M(3) receptors promotes release of LTB(4), suggesting that anti-muscarinic agents may be anti-inflammatory.
Background and purpose:
Macrophages release cytokines that may contribute to pulmonary inflammation in conditions such as chronic obstructive pulmonary disease. Thus, inhibition of macrophage ...cytokine production may have therapeutic benefit. p38 MAPK may regulate cytokine production, therefore, the effect of two p38 MAPK inhibitors, SB239063 and SD‐282, on the release of TNF‐α, GM‐CSF and IL‐8 from human macrophages was investigated.
Experimental approach:
Cytokine release was measured by ELISA. Immunoblots and mRNA expression studies were performed to confirm p38 MAPK isoform expression and activity. Macrophages were isolated from lung tissue of current smokers, ex‐smokers and emphysema patients and exposed to lipopolysaccharide. These cells then released cytokines in a concentration‐dependent manner.
Key results:
SB239063 only inhibited TNF‐α release (EC50 0.3±0.1 μM). Disease status had no effect on the efficacy of SB239063. SD‐282 inhibited both TNF‐α and GM‐CSF release from macrophages (EC50 6.1±1.4 nM and 1.8±0.6 μM respectively) but had no effect on IL‐8 release. In contrast, both inhibitors suppressed cytokine production in monocytes.
Conclusions and Implications:
The differential effects of p38 MAPK inhibitors between macrophages and monocytes could not be explained by differences in p38 MAPK isoform expression or activity. However, the stability of TNF‐α mRNA was significantly increased in macrophages compared to monocytes. These data suggest a differential involvement for p38 MAPK in macrophage cytokine production compared with monocytes. These effects are not due to lack of p38 activation or p38α expression in macrophages but may reflect differential effects on the stability of cytokine mRNA.
British Journal of Pharmacology (2006) 149, 393–404. doi:10.1038/sj.bjp.0706885
To assess the economic impact of inherited retinal disease (IRD) among Singaporeans.
IRD prevalence was calculated using population-based data. Focused surveys were conducted for sequentially ...enrolled IRD patients from a tertiary hospital. The IRD cohort was compared to the age- and gender-matched general population. Economic costs were expanded to the national IRD population to estimate productivity and healthcare costs.
National IRD caseload was 5202 cases (95% CI, 1734-11273). IRD patients (n = 95) had similar employment rates to the general population (67.4% vs. 70.7%; p = 0.479). Annual income was lower among IRD patients than the general population (SGD 19,500 vs. 27,161; p < 0.0001). Employed IRD patients had lower median income than the general population (SGD 39,000 vs. 52,650; p < 0.0001). Per capita cost of IRD was SGD 9382, with a national burden of SGD 48.8 million per year. Male gender (beta of SGD 6543, p = 0.003) and earlier onset (beta of SGD 150/year, p = 0.009) predicted productivity loss. Treatment of the most economically impacted 10% of IRD patients with an effective IRD therapy required initial treatment cost of less than SGD 250,000 (USD 188,000) for cost savings to be achieved within 20 years.
Employment rates among Singaporean IRD patients were the same as the general population, but patient income was significantly lower. Economic losses were driven in part by male patients with early age of onset. Direct healthcare costs contributed relatively little to the financial burden.
Ovarian clear cell carcinomas (OCCC) are a drug-resistant and aggressive type of epithelial ovarian cancer. We analyzed the molecular genetic profiles of OCCCs to determine whether distinct genomic ...subgroups of OCCCs exist.
Fifty pure primary OCCCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). Unsupervised hierarchical clustering using Ward's linkage analysis was performed to identify genomic subgroups of OCCCs. Survival analysis was performed using Kaplan-Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Differentially amplified regions between genomic subgroups of OCCCs were identified using a multi-Fisher's exact test.
Hierarchical cluster analysis revealed two distinct clusters of OCCCs with different clinical outcomes. Patients from cluster-1 had a significantly shorter median progression-free survival (PFS) than those from cluster-2 (11 vs. 65 months, P = 0.009), although estimates for ovarian cancer-specific survival (OCS) did not reach statistical significance (P = 0.065). In multivariate analysis, suboptimal debulking surgery and genomic cluster were independently prognostic for PFS. Recurrently amplified genomic regions with a significantly higher prevalence in cluster-1 than cluster-2 OCCCs were identified and validated. HER2 gene amplification and protein overexpression was observed in 14% of OCCCs, suggesting that this may constitute a potential therapeutic target for a subgroup of these tumors.
OCCCs constitute a heterogeneous disease at the genomic level despite having similar histological features. The pattern of genomic aberrations in subgroups of OCCCs is of clinical significance. We have identified recurrently amplified regions that may harbor potential therapeutic targets for subgroups of OCCCs.
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 ( K i 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 μM 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 (â¼2-11%) reduced the generation of tumor necrosis factor (TNF)α 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 âagedâ in culture medium. Under
this condition, BRL 50481 now inhibited TNFα generation in a concentration-dependent manner. In aged monocytes, rolipram,
Org 9935 (a PDE3 inhibitor), and prostaglandin E 2 inhibited TNFα 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.
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