Vera M. Keatings

Late response to allergen is associated with increased concentrations of tumor necrosis factor-α and IL-5 in induced sputum

Bronchial antigen challenge of sensitized atopic patients with asthma results in an early fall in FEV1, followed in a proportion of patients by a late (4 to 24 hours) fall. The late response is accompanied by an increase in bronchial reactivity, which is widely believed to reflect local influx and degranulation of inflammatory cells, particularly eosinophils, in association with elevated local secretion of cytokines. We hypothesized that the development of a late-phase bronchoconstrictor response and airway eosinophilia after allergen challenge of sensitized atopic patients with asthma is associated with elevated induced sputum concentrations of the eosinophil-active cytokines IL-5 and granulocyte-macrophage colony-stimulating factor and the proinflammatory cytokine tumor necrosis factor-alpha. We counted inflammatory leukocytes and measured cytokine concentrations in induced sputum at baseline and 24 hours after inhalational allergen challenge of 15 atopic patients with asthma who had previously demonstrated a late response. We observed significant increases in the numbers of eosinophils and the concentrations of their granule products, eosinophil cationic protein and eosinophil peroxidase. In contrast, the numbers of neutrophils and concentrations of two of their products, myeloperoxidase and human neutrophil lipocalin, did not significantly change. The numbers of sputum eosinophils correlated with the maximal late-phase fall in FEV1. Concentrations of IL-5 and tumor necrosis factor-alpha, but not granulocyte-macrophage colony-stimulating factor, were significantly elevated after allergen challenge. We conclude that the relatively noninvasive technique of induced sputum production can be used to monitor the effect of bronchial provocation on cytokine concentrations in asthma.

Association of MMP-12 polymorphisms with severe and very severe COPD: A case control study of MMPs - 1, 9 and 12 in a European population

BackgroundGenetic factors play a role in chronic obstructive pulmonary disease (COPD) but are poorly understood. A number of candidate genes have been proposed on the basis of the pathogenesis of COPD. These include the matrix metalloproteinase (MMP) genes which play a role in tissue remodelling and fit in with the protease - antiprotease imbalance theory for the cause of COPD. Previous genetic studies of MMPs in COPD have had inadequate coverage of the genes, and have reported conflicting associations of both single nucleotide polymorphisms (SNPs) and SNP haplotypes, plausibly due to under-powered studies.MethodsTo address these issues we genotyped 26 SNPs, providing comprehensive coverage of reported SNP variation, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations. We used logistic regression to adjust for age, gender, centre and smoking history.ResultsHaplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), showed an association with severe/very severe disease, corresponding to GOLD Stages III and IV.ConclusionsThose with the common A-A haplotype for these two SNPs were at greater risk of developing severe/very severe disease (p = 0.0039) while possession of the minor G variants at either SNP locus had a protective effect (adjusted odds ratio of 0.76; 95% CI 0.61 - 0.94). The A-A haplotype was also associated with significantly lower predicted FEV1 (42.62% versus 44.79%; p = 0.0129). This implicates haplotypes of MMP-12 as modifiers of disease severity.

Genetic variants of microsomal epoxide hydrolase and glutamate-cysteine ligase in COPD

The genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are poorly understood. Many candidate genes have been proposed, including enzymes that protect the lung against oxidative stress, such as microsomal epoxide hydrolase (EPHX1) and glutamate-cysteine ligase (GCL). To date, most reported findings have been for EPHX1, particularly in relation to functional variants associated with fast and slow metabolism of epoxide intermediates. The present study aimed to identify any association of variation in these genes with COPD susceptibility or severity. In total, 1,017 white COPD patients and 912 nondiseased age and sex matched smoking controls were genotyped for six single nucleotide polymorphisms (SNPs) in EPHX1 (including the fast and slow variants and associated haplotypes), and eight SNPs in the two genes encoding GCL. GCL is a rate-limiting enzyme in the synthesis of glutathione, a major contributor to anti-oxidant protection in the lung. No association of variation was found in EPHX1 or GCL with susceptibility to COPD or disease severity. This is the largest reported study to date and is well powered to detect associations that have been previously suggested. The current data indicate that these genetic variants are unlikely to be related to susceptibility or disease severity in white chronic obstructive pulmonary disease patients.

The SERPINE2 Gene and Chronic Obstructive Pulmonary Disease

To the Editor: In the February 2006 issue of the Journal, DeMeo et al.1 identified SERPINE2 as a positional candidate gene for susceptibility to chronic obstructive pulmonary disease (COPD [MIM 606963]) and reported on the association of polymorphic variants of this gene with early-onset disease in a family-based study and with severe disease in a case-control study. With early prior information provided by the authors, we have independently tested for an association of the SERPINE2 gene with COPD in the largest case-control study reported to date. Our study consists of 1,018 COPD cases and 911 controls prospectively recruited from six European centers. We have provided details about the patients elsewhere.2 The study population was screened for genotypes at the Medical Research Council (United Kingdom) Gene Services Unit for five SNPs (table 1) in the SERPINE2 gene. All the SNPs evaluated were reported in the study by DeMeo et al. as associated with disease, with three of the five associated with disease in both the family and case-control study cohorts they assessed. Table 1.  LD between SERPINE2 SNPs Expressed as r2[Note] We examined linkage disequilibrium (LD) between the SNPs (table 1) and evaluated SNP and haplotype associations as described elsewhere.2 DeMeo et al. did not report specific LD values between SNPs or noncontiguous SNPs contributing to haplotypes. SNPs and genotype frequencies in the study population are shown in table 2. We found no significant deviation from Hardy-Weinberg equilibrium in frequencies for any of the SNPs. Table 2.  SERPINE2 Genotype and Allele Frequencies in Controls and COPD Cases We found no association between any of the SERPINE2 SNPs and disease, in examining both the allelic and genotype distributions, although our study was well powered to detect associations of the magnitude observed by DeMeo et al., and we would have expected to see these frequency differences with the SNPs that we studied. We also failed to find a relationship between any haplotypes of these SNPs and disease (data not shown). It was of interest that the allele and genotype frequencies observed in our control and patient groups were virtually identical to those observed in control subjects by DeMeo et al., indicating a common distribution of SERPINE2 variants in the European and North American populations studied. Our previous study has also shown that there is no evidence of population stratification in our sample. Patients evaluated in both the family-based and case-control studies reported by DeMeo et al. represent a severe subset of the disease spectrum. To determine whether the association with SERPINE2 noted by DeMeo et al. was related to disease severity, we also analyzed SNP allele and genotype frequencies in the subgroup of our patients with forced expiratory volume at 1 s ⩽45% (n=388), a group that represents severe disease, but we failed to observe any association. Our inability to replicate the observations of DeMeo et al. in a more highly powered case-control study may be related to differences in the disease phenotype of the patients studied, because our patients included those with and without emphysema. The possibility, however, that the associations reported by DeMeo et al. represent false-positive results must also be considered. In this respect, it is of note that, in the study by DeMeo et al., different associations were reported for SNPs that are in linkage disequilibrium with one another. For example, rs3795879 and rs3795877 have an r2 value of 1 in HapMap, yet different associations with quantitative spirometric phenotypes were reported for the family study. Similarly, rs1438831 and rs920251 are in complete LD, with an r2 value of 1 in HapMap and 0.95 in our study; however, in DeMeo et al.’s case-control study, the allele and genotype frequencies of rs920251 were found to be significantly associated with disease (P values of 0.015 and 0.011, respectively), whereas no similar association was observed for rs1438831. In both instances, the almost complete linkage between these pairs of SNPs would be expected to result in similar associations. These results underline the importance of replication in other large independent studies before SERPINE2 can be unequivocally assigned as a candidate gene for COPD. It is becoming apparent that, to detect modest genetic effects for complex diseases, several independent studies may be required and the data may need to be subjected to meta-analysis. For example, this approach has been used to study Alzheimer disease (see Alzheimers Association Web site). Similar approaches need to be adopted for COPD. It would also be helpful to have similar criteria adapted for phenotypic selection and to plan prospective studies on this basis.

The role of IREB2 and transforming growth factor beta-1 genetic variants in COPD: a replication case-control study

BackgroundGenetic factors are known to contribute to COPD susceptibility and these factors are not fully understood. Conflicting results have been reported for many genetic studies of candidate genes based on their role in the disease. Genome-wide association studies in combination with expression profiling have identified a number of new candidates including IREB2. A meta-analysis has implicated transforming growth factor beta-1 (TGFbeta1) as a contributor to disease susceptibility.MethodsWe have examined previously reported associations in both genes in a collection of 1017 white COPD patients and 912 non-diseased smoking controls. Genotype information was obtained for seven SNPs in the IREB2 gene, and for four SNPs in the TGFbeta1 gene. Allele and genotype frequencies were compared between COPD cases and controls, and odds ratios were calculated. The analysis was adjusted for age, sex, smoking and centre, including interactions of age, sex and smoking with centre.ResultsOur data replicate the association of IREB2 SNPs in association with COPD for SNP rs2568494, rs2656069 and rs12593229 with respective adjusted p-values of 0.0018, 0.0039 and 0.0053. No significant associations were identified for TGFbeta1.ConclusionsThese studies have therefore confirmed that the IREB2 locus is a contributor to COPD susceptibility and suggests a new pathway in COPD pathogenesis invoking iron homeostasis.

Glucocorticoid treatment reduces exhaled nitric oxide in cystic fibrosis patients

In cystic fibrosis (CF), low concentrations of exhaled nitric oxide (NO) and reduced expression of inducible nitric oxide synthase (iNOS) in airway epithelium have been reported. However, abundant iNOS expression has been found in the subepithelial tissues and elevated concentrations of NO metabolites in breath condensate and sputum. These conflicting results may be explained by increased scavenging of NO by superoxide radicals, resulting in rapid conversion to peroxynitrite, so that only a small proportion of the NO produced in the lung tissue reaches the airway lumen. If iNOS were active in the CF lung, exhaled NO would be further reduced by glucocorticoid treatment. CF patients (n = 13) were recruited to a double-blind, placebo-controlled study with crossover. Treatment comprised prednisolone or placebo for 5 days with a 9 day washout. After each treatment, exhaled NO was measured, spirometry performed and blood collected for measurement of serum nitrogen dioxide/nitrous oxide (NO2/NO3). Ten patients (8 male) completed the study. Following prednisolone treatment (mean +/- SD) exhaled NO concentration (3.1 +/- 1.6 parts per billion (ppb)) was significantly reduced versus placebo treatment (4.9 +/- 4.2 ppb; p<0.05, Wilcoxon signed-rank test). Spirometric indices and serum NO2/NO3 concentration were unchanged. These findings support the hypothesis that glucocorticoids suppress nitric oxide production in cystic fibrosis airways by reducing inducible nitric oxide synthase expression or by inhibiting recruitment of neutrophils, cells which express inducible nitric oxide synthase.

Variation in the tumour necrosis factor gene is not associated with susceptibility to COPD

To the Editors: In a recent issue of the European Respiratory Journal , Tanaka et al. 1 studied polymorphisms in the tumour necrosis factor (TNF) and lymphotoxin A genes with respect to their effect on lung function of smokers, and failed to find any association with chronic obstructive pulmonary disease (COPD) phenotypes. Tanaka et al. 1 acknowledge that their work is not a true case–control study, but that it would be better described as an investigation of genetic contribution to disease severity. There have been several studies of variation in TNF with respect to susceptibility to COPD, although many of these have used relatively small sample sizes and are therefore underpowered, and so are likely to lead to results that cannot be replicated. As part of a European Union collaborative project, we have …

Induced sputum and BAL analysis in COPD

An essential component of airways in flammation research is the examination of airway luminal fluid. Since its development as a sampling technique over 20 years ago, bronchoalveolar lavage (BAL) has become the most widely used method of evaluation of distal airway and alveolar inflammation. The widespread use of BAL reflects its relative ease and lack of clinical complications, particularly in comparison to airway or lung biopsy. However, while BAL is now a universally used tool in research and diagnosis of interstitial lung diseases (ILDs) its use in evaluating airway inflammation in COPD is less common. The lesser use of BAL in COPD studies reflects a lower level of tolerance of the procedure in patients with moderate to severe airway obstruction and greater difficulty in obtaining adequate samples from these same patient groups [1, 2].