Genetic variants of human beta-defensin-1 and chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is due to interactions between cigarette smoke exposure and other risk factors. Genetic variations of human beta-defensin-1 (hBD-1), an endogenous antimicrobial peptide in the airway, were investigated in 60 patients and 213 healthy volunteers by single-strand conformation and restriction fragment length polymorphism analysis and DNA sequencing. Four nucleotide variations in the 5' and 3' untranslated regions and two nonsynonymous substitutions in the coding region were identified. Of these, a newly found Ile38 variant was observed in 15.0% of patients but only in 2.8% of healthy individuals and was significantly associated with the disease (OR = 6.1, 95% confidence intervals 2.0-8.3, P = 0.0012). More than 80% of those with Ile38 experienced sputum production for more than 3 months during the follow-up period. Genetic variations in hBD-1 may define a high-risk subgroup of COPD where the component of chronic bronchitis is predominant.     

The association of monocyte chemotactic protein-1 and CC chemokine receptor 2 gene variants with chronic obstructive pulmonary disease

Chemokines are potent proinflammatory cytokines that are implicated in numerous inflammatory diseases. Monocyte chemoattractant protein-1 (MCP-1) and its receptor CC chemokine receptor-2 (CCR2) play a major role in the recruitment of inflammatory cells to the lungs of patients with chronic obstructive pulmonary disease (COPD). We investigated a possible association between polymorphisms in MCP-1 and CCR2 genes (MCP-1 -2518 A/G and CCR2 190G/A or V64I) and the development of COPD. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 386 COPD cases and 398 age-matched healthy controls. Frequency of MCP-1 2518GG genotype for cases and controls was 0.396 and 0.324, respectively; individuals who had the GG genotype had a 1.59-fold increased risk of COPD (p=0.036). Frequency of CCR2 190AA (64I/64I) genotype for cases and controls was 0.285 and 0.21, respectively; subjects carrying the 64I/64I genotype had a 2.04-fold increased risk of COPD compared with the wild-type genotype (p=0.001). When analyzing the allele combination of these two polymorphisms, the combinations MCP-1-A/CCR2-A and MCP-1-G/CCR2-A were detected in significantly higher numbers in COPD cases than in healthy controls (odds ratio [OR]=1.50, 95% confidence interval [CI]: 1.04-2.17, p=0.032; and OR=1.89, 95% CI: 1.38-2.60, p=7.38×10(-5)). These data suggest that MCP-1 -2518 A/G and CCR2 190G/A polymorphisms are new risk factors for COPD.     

The bactericidal effect of human secreted group IID phospholipase A2 results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca(2+)-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10μg/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca(2+)-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca(2+)-independent damaging activity may play a role in the bactericidal function of the protein.     

Analysis of PGC-1alpha variants Gly482Ser and Thr612Met concerning their PPARgamma2-coactivation function

Based on protein sequence homology searches, we found a conserved open reading frame within the genome of several human pathogenic bacteria showing a resemblance to the mammalian TIR domain. We cloned, expressed, and characterized the corresponding gene product from Paracoccus denitrificans using several biophysical techniques. The protein consists of two independently folded domains. As predicted from the amino acid sequence and experimentally confirmed here, the N-terminal domain consists of a alpha-helical coiled-coil. The NMR data indicates that the C-terminal TIR-like domain folds into a compact protein. Finally, using GST pull-down experiments, we show that the bacteria TIR-like domain binds to the mammalian receptor (TLR4) and adaptor (MyD88) TIR domains. We postulate that prokaryotic pathogens utilize the TIR-like proteins to interfere with the innate immune response of the mammalian host so that the bacterial infection can progress undetected.     

Inhaled adenosine A(2A) receptor agonists for the treatment of chronic obstructive pulmonary disease

COPD is a major cause of mortality in the western world. A(2A) agonists are postulated to reduce the lung inflammation that causes COPD. The cardiovascular effects of A(2A) agonists dictate that a compound needs to be delivered by inhalation to be therapeutically useful. A strategy of minimizing side-effect liability by maximizing systemic clearance was followed and pharmacological and pharmacokinetic SAR of a series of inhaled A(2A) agonists described. A sevenfold improvement in potency and 150-fold reduction in side-effect liability over the lead compound CGS-21680, were obtained.     

Genetic analysis of IREB2, FAM13A and XRCC5 variants in Chinese Han patients with chronic obstructive pulmonary disease

Recently, variants (rs2568494, rs2869967 and rs3821104) in the IREB2, FAM13A and XRCC5 genes were found to be associated with chronic obstructive pulmonary disease (COPD) in non-Asian populations by genome-wide association study (GWAS) analysis. To evaluate whether variants in these genes are related to COPD in Chinese Han population, we investigated COPD patients of Chinese Han ethnicity from Mainland China. Significant differences in genotypic distributions (χ² = 6.319, p = 0.042 for rs2869967; χ² = 6.062, p = 0.048 for rs3821104) and allele distributions (χ² = 4.014, p = 0.045 for rs2869967; χ² = 5.607, p = 0.018 for rs3821104) were observed between patients and control subjects for variants rs2869967 and rs3821104, whereas no statistically significant associations for genotypic and allelic distribution between IREB2 rs2568494 and COPD phenotype (p > 0.05) were identified. Our results support that FAM13A rs2869967 and XRCC5 rs3821104 are associated with COPD in Chinese Han population.     

Structural and phylogenetic basis for the classification of group III phospholipase A2

Secretory phospholipase A₂ (PLA₂) catalyses the hydrolysis of the sn-2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that are known mediators of inflammation. The group III PLA₂ enzymes are present in a wide array of organisms across many species with completely different functions. A detailed understanding of the structure and evolutionary proximity amongst the enzymes was carried out for a meaningful classification of this group. Fifty protein sequences from different species of the group were considered for a detailed sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific ‘amino acid signatures’. Structural analysis reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C-terminal domain and the quaternary conformational state. Character and distance based phylogenetic analysis of these sequences are in accordance with the structural features. The outcomes of the structural and phylogenetic analysis lays a convincing platform for the classification the group III PLA₂s into (1A) venomous insects; (IB) non-venomous insects; (II) mammals; (IIIA) gila monsters; (IIIB) reptiles, amphibians, fishes, sea anemones and liver fluke, and (IV) scorpions. This classification also helps to understand structure-function relationship, enzyme-substrate specificity and designing of potent inhibitors against the drug target isoforms.     

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis.

Methods

The expression of TLR-2, TLR-4 and CD14 in monocytes was analyzed by flow cytometry. To study the functional responses of these receptors, monocytes were stimulated with peptidoglycan or lipopolysaccharide and the amounts of TNFα and IL-6 secreted were determined by ELISA.

Results

We found that the expression of TLR-2 was up-regulated in peripheral blood monocytes from COPD patients, either clinically stable or during AECOPD, as compared to never smokers or smokers with normal lung function. Upon stimulation with TLR-2 ligand monocytes from COPD patients secreted increased amounts of cytokines than similarly stimulated monocytes from never smokers and smokers. In contrast, the expressions of TLR-4 and CD14 were not significantly different between groups, and the response to lipopolysaccharide (a TLR-4 ligand) stimulation was not significantly different either. At discharge from hospital TLR-2 expression was down-regulated in peripheral blood monocytes from AECOPD patients. This could be due to the treatment with systemic steroids because, in vitro, steroids down-regulated TLR-2 expression in a dose-dependent manner. Finally, we demonstrated that IL-6, whose plasma levels are elevated in patients, up-regulated in vitro TLR-2 expression in monocytes from never smokers.

Conclusion

Our results reveal abnormalities in TLRs expression in COPD patients and highlight its potential relationship with systemic inflammation in these patients.

     

Molecular basis of ligand recognition and activation of human V2 vasopressin receptor

Dear Editor, Vasopressin type 2 receptor(V2R)belongs to the vasopressin(VP)/oxytocin(OT)receptor subfamily of G protein-coupled receptors(GPCRs),which comprises...     

Cellular distribution, post-translational modification, and tumorigenic potential of human group III secreted phospholipase A(2)

Human group III secreted phospholipase A(2) (sPLA(2)-III) consists of a central group III sPLA(2) domain flanked by unique N- and C-terminal domains. We found that the sPLA(2) domain alone was sufficient for its catalytic activity and for its prostaglandin E(2) (PGE(2))-generating functions in various cell types. In several if not all cell types, the N- and C-terminal domains of sPLA(2)-III were proteolytically removed, leading to the production of the form containing only the sPLA(2) domain, which could be further N-glycosylated at two consensus sites. Immunohistochemistry demonstrated that sPLA(2)-III was preferentially expressed in the microvascular endothelium in human tissues with inflammation, ischemic injury, and cancer. In support of this, sPLA(2)-III was induced in cultured microvascular endothelial cells after stimulation with proinflammatory cytokines. Expression of sPLA(2)-III was also associated with various tumor cells, and colorectal cancer cells transfected with sPLA(2)-III exhibited enhanced PGE(2) production and cell proliferation, which required sPLA(2)-III catalytic activity. When implanted into nude mice, the sPLA(2)-III-transfected cells formed larger solid tumors with increased angiogenesis compared with control cells. Moreover, small interfering RNA for sPLA(2)-III significantly reduced PGE(2) production and proliferation of colorectal cancer cells. Taken together, these results reveal unique cell type-specific processing and N-glycosylation of sPLA(2)-III and the potential role of this enzyme in cancer development by stimulating tumor cell growth and angiogenesis.     

Significance of P2X7 receptor variants to human health and disease

The human P2X7 receptor is a trimeric ligand-gated cation channel coded by the P2XR7 gene located at chromosome position 12q24. P2X7 is expressed in a wide variety of normal and disease-associated cell types. Activation of this receptor by extracellular adenosine 5'-triphosphate results in numerous downstream events including the release of pro-inflammatory mediators, cell proliferation or death, and killing of intracellular pathogens. As a result, P2X7 plays important roles in inflammation, immunity, bone homeostasis, neurological function and neoplasia. The P2XR7 gene encodes a P2X7 subunit 595 amino acids in length, however splice isoforms that can alter receptor expression and function, and modify the signaling properties downstream of receptor activation also exist. Moreover, the relative amount of P2X7 function varies between human individuals due to numerous single nucleotide polymorphisms resulting in either loss- or gain-of-function. Combinations of these polymorphisms give rise to various haplotypes that can also modify P2X7 function. Collectively, P2X7, and its splice and polymorphic variants are attracting considerable interest in relation to human health and disease, including the development and publication of a number of patents.     

Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A₂ (sPLA₂s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A₂ (hsPLA₂-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA₂-IID in Escherichia coli, the DNA-coding sequence for hsPLA₂-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA₂-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A₂. The refolded recombinant hsPLA₂-IID demonstrated Ca²⁺-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA₂-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.     

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2)

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.     

A clonal group of nontypeable Haemophilus influenzae with two IgA proteases is adapted to infection in chronic obstructive pulmonary disease

Strains of nontypeable Haemophilus influenzae show enormous genetic heterogeneity and display differential virulence potential in different clinical settings. The igaB gene, which encodes a newly identified IgA protease, is more likely to be present in the genome of COPD strains of H. influenzae than in otitis media strains. Analysis of igaB and surrounding sequences in the present study showed that H. influenzae likely acquired igaB from Neisseria meningitidis and that the acquisition was accompanied by a ~20 kb genomic inversion that is present only in strains that have igaB. As part of a long running prospective study of COPD, molecular typing of H. influenzae strains identified a clonally related group of strains, a surprising observation given the genetic heterogeneity that characterizes strains of nontypeable H. influenzae. Analysis of strains by 5 independent methods (polyacrylamide gel electrophoresis, multilocus sequence typing, igaB gene sequences, P2 gene sequences, pulsed field gel electrophoresis) established the clonal relationship among the strains. Analysis of 134 independent strains collected prospectively from a cohort of adults with COPD demonstrated that ~10% belonged to the clonal group. We conclude that a clonally related group of strains of nontypeable H. influenzae that has two IgA1 protease genes (iga and igaB) is adapted for colonization and infection in COPD. This observation has important implications in understanding population dynamics of H. influenzae in human infection and in understanding virulence mechanisms specifically in the setting of COPD.     

The effect of CYP3A4 genetic variants on the susceptibility to chronic obstructive pulmonary disease in the Hainan Han population

PurposeGenetic polymorphisms act a crucial role in chronic obstructive pulmonary disease (COPD) progression. This study aimed to investigate the correlation between CYP3A4 variants and COPD risk.MethodsWe carried out a case-control study of 821 individuals (313 patients and 508 healthy subjects) to identify the correlation of CYP3A4 SNPs with COPD risk in the Hainan Han population. The association was evaluated by Odds ratios (OR) and 95% confidence intervals (CI).ResultsOur study showed that rs4646437 polymorphism was related to a significantly increased susceptibility to COPD (OR 1.45, 95% CI = 1.10–1.90, p = 0.008). Stratified analyses indicated that rs4646437 polymorphism was significantly related to an increased risk of COPD in males (OR 1.95, 95% CI = 1.19–3.20, p = 0.008). However, rs4646440 played a protective role in females (OR 0.54, 95% CI = 0.31–0.93, p = 0.024). Rs4646437 was found to significantly improve the risk of COPD in smokers (OR 1.67, 95% CI = 1.12–2.48, p = 0.011). While rs4646440 had a significantly lower susceptibility to COPD in non-smokers (OR 0.64, 95% CI = 0.45–0.90, p = 0.010). Haplotype analysis revealed that A_rs4646440T_rs35564277 haplotype of CYP3A4 was found to increase the risk of COPD in non-smokers (OR 1.71, 95% CI = 1.04–2.82, p = 0.034).ConclusionOur result gives a new understanding of the association between CYP3A4 gene and COPD in the Hainan Han population.     

IL-17A对慢性阻塞性肺疾病的干预作用及其机制*

目的:探讨白细胞介素-17A(IL-17A)对慢性阻塞性肺疾病(COPD)的干预作用及其机制。方法:C57BL/6小鼠随机分为野生型空白对照组、野生型COPD组和IL-7A敲除COPD组,每组20只。野生型空白对照组小鼠不做任何处理,其余两组小鼠暴露于香烟烟雾(1支/次,4次/日,每次45 min,每次间隔时间为1 h,总干预时间为90 d)制作COPD模型。干预结束24 h后,利用动物肺功能检测系统测定小鼠肺功能。收集小鼠支气管肺泡灌洗液(BALF),测定BALF细胞计数和分类。收集小鼠肺组织,采用流式细胞法测定气道上皮IL-17A表达水平,采用酶联免疫吸附法测定肺组织炎症因子水平。采用蛋白免疫印迹法测定小鼠肺组织JNK/AP1信号通路蛋白表达水平。结果:与野生型空白对照组小鼠比较,野生型COPD组小鼠气道上皮IL-17A表达水平明显升高,吸气峰流速(PIF)和呼气峰流速(PEF)明显降低,BALF中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞数明显升高,肺组织CXC类趋化因子1(CXCL1)、CXC类趋化因子2(CXCL2)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)表达水平明显升高,JNK、cJun和cFos磷酸化水平及AP1表达水平明显升高(P＜0.05);与野生型COPD组小鼠比较,IL-7A敲除COPD组小鼠气道上皮IL-17A表达水平明显降低,PIF和PEF明显升高,BALF中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞数明显降低,肺组织CXCL1、CXCL2、IL-1β和IL-6表达水平明显降低,JNK、cJun和cFos磷酸化水平及AP1表达水平明显降低(P＜0.05)。结论:香烟烟雾可诱导小鼠气道上皮产生IL-17A,降低(或抑制)IL-17A的产生(或表达或分泌),通过抑制JNK/AP1信号通路,减轻COPD气道炎症反应,改善COPD小鼠肺功能。     

Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2

Phospholipases A2 (PLA2s) play a key role in inflammatory processes through production of precursors of eicosanoids and platelet-activating factor. Recently, we described the purification of a novel approximately 100-kDa cytosolic PLA2 (cPLA2) from human monoblast U937 cells that is activated by physiological (intracellular) concentrations of Ca2+ (Kramer, R. M., Roberts, E. F., Manetta, J., and Putnam, J. E. (1991) J. Biol. Chem. 266, 5268-5272). Here we report the isolation of the complementary DNA encoding human cPLA2 and confirm its identity by expression in bacteria and in hamster cells. The predicted 749-amino acid cPLA2 protein has no similarity to the well known secretory PLA2s, but contains a structural element homologous to the C2 region of protein kinase C. The molecular cloning of cPLA2 will allow further studies defining the structure, function, and regulation of this novel PLA2.     

The ion channel transient receptor potential melastatin-2 does not play a role in inflammatory mouse models of chronic obstructive pulmonary diseases

Background

There is strong evidence that oxidative stress is associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). The transient receptor potential melastatin-2 (TRPM2) is an oxidative stress sensing channel that is expressed in a number of inflammatory cells and therefore it has been suggested that inhibition of TRPM2 could lead to a beneficial effect in COPD patients. In this study, we have investigated the role of TRPM2 in a variety of mouse models of oxidative stress and COPD using TRPM2-deficent mice.

Methods

Mice were exposed to ozone (3 ppm for 4 h) or lipopolysaccharide (LPS, 0.3 mg/kg, intranasaly). In another model, mice were exposed to tobacco smoke (750 μg/l total wet particulate matter) for 30 min twice a day on three consecutive days. For the exacerbation model, the smoke exposure on the morning of day 3 animals was replaced with intranasal administration of LPS (0.3 mg/kg). Animals were killed 3 and 24 h after the challenge (ozone and LPS model) or 18 h after the last tobacco smoke exposure. In vitro neutrophil chemotaxis and monocyte activation were also studied using cells isolated from wild type and TRPM2-deficient animals. Statistical significance for the in vivo data (P < 0.05) was determined using analysis of variance with Kruskal-Wallis and Dunns multiple comparison test.

Results

In all models studied, no difference in the bronchoalveolar lavage inflammation could be evidenced when comparing wild type and TRPM2-deficient mice. In addition, no difference could be seen in the lung inflammation as assessed by the measurement of various cytokines/chemokines. Similarly in various in vitro cellular activation assays using isolated neutrophils and monocytes no significant differences could be observed when comparing wild type and TRPM2-deficient mice.

Discussion

We have shown, in all the models tested, no difference in the development of airway inflammation or cell activation between TRPM2-deficient mice and their wild type counterparts. These results would suggest that inhibiting TRPM2 activity in COPD would have no anti-inflammatory effect.