Validation of assays for inflammatory mediators in exhaled breath condensate.

The use of exhaled breath condensate (EBC) as a tool for noninvasive assessment of lung inflammation is becoming commonplace. Many authors use commercial ELISA kits to measure inflammatory mediators within EBC. However, the very low concentrations of mediators within EBC are often below the commercially validated concentration range of the relevant ELISA and crucially below the linear part of the sigmoid standard curve. The present study seeks to validate a series of assays for use in EBC and to compare the results in EBC with those from matched sol phase sputum samples. The following mediators were measured by ELISA: leukotriene (LT)B(4), interleukin (IL)-8, secretory leukoprotease inhibitor and alpha(1)-antitrypsin (AAT). Myeloperoxidase was measured by chromogenic substrate assay. Mediator concentrations reached the lower limit of quantification in only one assay (AAT) in 19.6% of subjects, while mediator concentrations reached the lower limit of detection in three assays (LTB(4), IL-8 and AAT in 31, 6.5 and 61% of subjects, respectively). No significant correlations were present between any mediators in EBC and sol phase sputum. The results of the present study indicate that care must be exercised when interpreting mediator measurements in exhaled breath condensate and that assays must be validated at concentrations relevant to those found within the biological fluid.

Inter-relationships between inflammatory markers in patients with stable COPD with bronchitis: intra-patient and inter-patient variability.

BACKGROUND: Measurements of pulmonary biomarkers can be used to monitor airway inflammation in chronic obstructive pulmonary disease (COPD), but the variability of sampled biomarkers and their inter-relationships are poorly understood. A study was undertaken to examine the intra- and inter-patient variability in spontaneous sputum samples from patients in the stable state and to describe the relationship between biomarkers, cell counts and markers of disease. METHODS: Sputum interleukin-1beta, tumour necrosis factor alpha, interleukin 8, myeloperoxidase, leucotriene B4, growth-related oncogene alpha and differential cell counts were measured in patients with moderate to severe stable COPD (n = 14) on 11 occasions over a 1-month period. RESULTS: There was significant variability in all inflammatory indices (median intra-patient coefficient of variation (CV) 35% (IQR 22-69), median inter-patient CV 102% (IQR 61-145)). Variability could be reduced by using a rolling mean of individual patient data points. Sample size calculations were undertaken to determine the number of patients required to detect a 50% reduction in neutrophil count. Using a crossover design of a putative effective treatment, the number needed using one data point per patient was 72, reducing to 23 when a mean of three data points was used. Significant correlations were demonstrated both between the inflammatory biomarkers themselves and between inflammatory biomarkers and markers of disease. Some relationships were not apparent when results from a single sample were used. The reliability of inter-relationships improved as more data points were used for each patient. CONCLUSIONS: Clear relationships exist between inflammatory biomarkers in patients with stable COPD. Sequential sampling reduced the variability of individual mediators and the potential number of patients needed to power proof of concept interventional studies.

Antiplatelet drug use in a diabetic clinic.

BACKGROUND: There are definite indications for antiplatelet therapy in diabetes in the presence of large-vessel disease, but in the absence of large-vessel disease, the evidence is less clear. There is also evidence that antiplatelet therapy is under-prescribed. AIM: To investigate the use of antiplatelet drugs in patients attending a diabetic clinic in a large teaching hospital. DESIGN: Retrospective case-note survey. METHODS: We examined the case-notes of 300 consecutive diabetic patients, to determine whether antiplatelet therapy was being used in appropriate patients, including those with established large-vessel disease, hypertension and nephropathy or microalbuminuria. RESULTS: The patients were of mean +/- SD age 61 +/- 13 years, diabetes duration 10 +/- 8 years, BMI 31.4 +/- 6.7 kg/m(2) and HbA(1c) 8.3 +/- 1.5%; 276 (92%) had type 2 diabetes, and 162 (54%) were male. Antiplatelet drugs were being taken by 157 (52%) who fulfilled the survey standard for treatment; a further 83 (28%) met the survey standard but were not receiving treatment, of whom 48 (16% of the total group) had no valid contraindication. DISCUSSION: A significant minority of diabetic patients are being denied antiplatelet drugs despite good indications.

Endothelial interactions of neutrophils under flow in chronic obstructive pulmonary disease.

It is generally accepted that the neutrophil is central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Enhanced endothelial interactions of this cell may contribute to the susceptibility of smokers who develop the disease; however, these interactions have not previously been studied in COPD. The aim of the current study was to determine whether enhanced endothelial interactions of neutrophils from smokers are a predisposing factor for the development of COPD. Endothelial interactions under flow and adhesion molecule expression of peripheral blood neutrophils were compared between seven never-smokers (NS), seven healthy smokers (HS), 11 COPD patients with severe alpha1-antitrypsin deficiency (PiZ) and neutrophils from 11 COPD patients without the deficiency (PiM). Total adhesive and migratory responses (per mm2 endothelium per 10(6) neutrophils) were significantly greater in the PiM group (mean+/-se 704.2+/-57.9 versus 509.3+/-48.8 in the PiZ group, 499.3+/-40.1 in the HS and 491.2+/-33.7 in the NS). This corresponded with increased macrophage antigen-1 (CD11b) expression on stimulated neutrophils in the PiM group compared with the PiZ group (mean+/-se relative fluorescence intensity 1.4+/-0.1 versus 1.1+/-0.1). In conclusion, the enhanced endothelial interaction of neutrophils from smokers who have developed chronic obstructive pulmonary disease in the presence of normal levels of alpha1-antitrypsin deficiency, but not in those with severe alpha(1)-antitrypsin deficiency, suggests that this is a predisposing factor for the development of the disease, and upregulation of macrophage antigen-1 may be responsible.

Proteolytic activity of the house dust mite allergen Der p 1 enhances allergenicity in a mouse inhalation model.

BACKGROUND: We have recently demonstrated that intraperitoneal immunization of mice with proteolytically active Der p 1, the major house dust mite allergen, results in a significant and selective enhancement of total and Der p 1-specific IgE synthesis compared to mice immunized with proteolytically inactive Der p 1. OBJECTIVE: To investigate whether the proteolytic activity of Der p 1 would lead to enhanced inflammatory cellular infiltration of the lungs and systemic IgE production when administered through the respiratory system, which is the natural route of entry for this allergen. METHODS: Groups of mice were initially sensitized with proteolytically active Der p 1 through the intraperitoneal and the subcutaneous routes and subsequently exposed intranasally to either proteolytically active Der p 1, inactive Der p 1 or PBS. The extent of cellular infiltration of the lungs and systemic IgE production in the three animal groups were then compared. RESULTS: Here, we show for the first time that the administration of proteolytically active Der p 1 to mice through the intranasal route leads to significant inflammatory cellular infiltration of the lungs and systemic production of IgE. CONCLUSIONS: These data underline the important role of the proteolytic activity of Der p 1 in driving the allergic response in the lungs.

Resolution of bronchial inflammation is related to bacterial eradication following treatment of exacerbations of chronic bronchitis.

BACKGROUND: Recent studies of the role of bacteria in chronic bronchitis have shown that bacterial colonisation is associated with enhanced inflammation and that purulent acute exacerbations of chronic bronchitis (AECB) are associated with bacteria and characterised by increased inflammation. Changes in bronchial inflammation in response to the success or failure of bacterial eradication following AECB were therefore studied. METHODS: Bacterial quantitative culture and sputum markers of inflammation (myeloperoxidase (MPO), neutrophil elastase, leukotriene B4 (LTB4), sol:serum albumin ratio, and secretory leukoprotease inhibitor) were measured in patients presenting with culture positive purulent AECB and repeated 10 days and 2 months later. 41 patients provided sputum sufficient for both bacteriology and assessment of inflammation at baseline and day 10, and 46 provided sufficient sample for bacteriology, 40 of which could also be analysed for inflammation at 2 months (when clinically stable). RESULTS: At day 10, 17 of the 41 patient samples had a positive bacterial culture. In the stable state, 18 of the 46 samples had a positive culture, but with a significantly lower bacterial load than at presentation. Although there was no difference between the groups at presentation, the concentration of MPO was lower (p<0.05) in those in whom bacteria were eradicated by day 10 than in those with persisting bacteria. The LTB4 concentration was similarly lower (p<0.001) in those in whom bacteria were eradicated than in those with persistent bacteria. In the stable clinical state the concentrations of both MPO and LTB4 were lower in those in whom bacteria were eradicated than in patients with persisting bacteria. CONCLUSION: Resolution of bronchial inflammation following AECB is related to bacterial eradication. Those in whom bacteria continue to be cultured in their sputum have partial resolution of inflammation which may reflect continued stimulation by the reduced bacterial load.

Effect of sputum processing with dithiothreitol on the detection of inflammatory mediators in chronic bronchitis and bronchiectasis.

BACKGROUND: Sputum analysis is used increasingly to assess airway inflammation in patients with chronic obstructive pulmonary disease, including those with chronic bronchitis and bronchiectasis. However, it is not known whether dithiothreitol (DTT), a reducing mucolytic agent regularly used to homogenise sputum, affects the detection of inflammatory mediators in the sputum soluble phase from such patients. METHODS: Thirty two spontaneous sputum samples were collected from 13 patients with chronic bronchitis and 17 with bronchiectasis. An aliquot from each sample was treated with either freshly prepared 0.1% DTT plus normal saline (NaCl) or NaCl alone, then ultracentrifuged to obtain the sputum sol phase. Interleukin (IL)-1beta, IL-6, IL-8, leukotriene B(4) (LTB(4)), secretory leukoprotease inhibitor (SLPI), alpha-1-antitrypsin (alpha(1)-AT), and tumour necrosis factor alpha (TNFalpha) were measured by ELISA, and neutrophil elastase (NE) and myeloperoxidase (MPO) by chromogenic substrate assay. The effect of DTT on the detection of assay standards was also determined. RESULTS: Median levels of IL-1beta, IL-6, IL-8, SLPI, and NE were similar in the DTT and NaCl treated samples. There was a significant reduction in median (IQR) levels of detectable TNFalpha (0.07 (0.00-0.47) pM v 0.90 (0.06-6.98) pM, p<0.001), LTB(4) (1.67 (1.31-2.64) nM v 2.29 (0.95-4.22) nM, p<0.05) and MPO (0.00 (0.00-0.00) mg/l v 4.48 (0.00-33.66) mg/l, p<0.001) and a small increase in the median alpha(1)-AT concentration (0.05 (0.03-0.08) nM v 0.03 (0.02-0.08) nM, p<0.01) in the DTT treated samples. DTT had no effect on the assay standards for IL-1beta, IL-8 or TNFalpha, but at higher concentrations it did affect IL-6, SLPI, NE, and LTB(4) standards (43%, 70%, 76% and 643% of control value for top standard, respectively) and at all concentrations DTT completely abolished MPO activity. CONCLUSIONS: Sputum processing with DTT significantly reduces the detectable concentration of TNFalpha, LTB(4) and MPO, and produces a small but significant increase in median alpha(1)-AT levels. To avoid this problem we recommend that an untreated aliquot of sputum be retained for cytokine analysis, unless the assay has been specifically validated.

Sputum chemotactic activity in chronic obstructive pulmonary disease: effect of alpha(1)-antitrypsin deficiency and the role of leukotriene B(4) and interleukin 8.

BACKGROUND: Neutrophil recruitment to the airway is thought to be an important component of continuing inflammation and progression of chronic obstructive pulmonary disease (COPD), particularly in the presence of severe alpha(1)-antitrypsin (alpha(1)-AT) deficiency. However, the chemoattractant nature of secretions from these patients has yet to be clarified. METHODS: The chemotactic activity of spontaneous sputum from patients with stable COPD, with (n=11) and without (n=11) alpha(1)-AT deficiency (PiZ), was assessed using the under-agarose assay. The contribution of leukotriene B(4) (LTB(4)) and interleukin 8 (IL-8) to the chemotactic activity was examined using an LTB(4) receptor antagonist (BIIL 315 ZW) and an IL-8 monoclonal antibody, respectively. RESULTS: Sputum neutrophil chemotactic activity (expressed as % n-formylmethionyl leucylphenylalanine (fMLP) control) was significantly higher in patients with alpha(1)-AT deficiency (mean (SE) 63.4 (8.9)% v 36.7 (5.5)%; mean difference 26.7% (95% CI 4.9 to 48.4), p<0.05). The mean (SE) contribution of both LTB(4) and IL-8 (expressed as % fMLP control) was also significantly higher in alpha(1)-AT deficient patients than in patients with COPD with normal levels of alpha(1)-AT (LTB(4): 31.9 (6.3)% v 18.0 (3.7)%; mean difference 13.9% (95% CI -1.4 to 29.1), p<0.05; IL-8: 24.1 (5.2)% v 8.1 (1.2)%; mean difference 15.9% (95% CI 4.7 to 27.2), p<0.05). When all the subjects were considered together the mean (SE) contribution of LTB(4) (expressed as % total chemotactic activity) was significantly higher than IL-8 (46.8 (3.5)% v 30.8 (4.6)%; mean difference 16.0% (95% CI 2.9 to 29.2), p<0.05). This difference was not significantly influenced by alpha(1)-AT phenotype (p=0.606). CONCLUSIONS: These results suggest that the bronchial secretions of COPD patients with alpha(1)-AT deficiency have increased neutrophil chemotactic activity. This relates to the increased levels of IL-8 and, in particular LTB(4), which accounted most of the sputum chemotactic activity in the patients with COPD as a whole. Increased chemotactic activity, together with inhibitor deficiency, may contribute to the more rapid disease progression seen in alpha(1)-AT deficiency via increased neutrophil recruitment and release of neutrophil elastase.

Changes in bronchial inflammation during acute exacerbations of chronic bronchitis.

There are little data describing noncellular changes in bronchial inflammation during exacerbations of chronic bronchitis. The relationship between sputum colour and airway inflammation at presentation has been assessed during an exacerbation in patients with chronic bronchitis and a primary care diagnosis of chronic obstructive pulmonary disease. Sputum myeloperoxidase, neutrophil elastase, leukotriene B4 (LTB4), interleukin-8 (IL-8), sol:serum albumin ratio and serum C-reactive protein were measured in patients presenting with an exacerbation and mucoid (n = 27) or purulent sputum (n = 42). Mucoid exacerbations were associated with little bronchial or systemic inflammation at presentation, and sputum bacteriology was similar to that obtained in the stable state. Purulent exacerbations were associated with marked bronchial and systemic inflammation (p < 0.025 for all features) and positive sputum cultures (90%). Resolution was related to a significant reduction in LTB4 (p < 0.01), but no change in IL-8, suggesting that LTB4 may be more important in neutrophil recruitment in these mild, purulent exacerbations. In the stable state, IL-8 remained higher in patients who had experienced a purulent exacerbation (2p < 0.02). The presented results indicate that exacerbations of chronic bronchitis, defined by sputum colour, differ in the degree of bronchial and systemic inflammation. Purulent exacerbations are related to bacterial infection, and are associated with increased neutrophilic inflammation and increased leukotriene B4 concentrations.

Assessment of airway neutrophils by sputum colour: correlation with airways inflammation.

BACKGROUND: Airway inflammation, with recruitment of neutrophils to the airway lumen, results in purulent secretions and a variety of potential adverse consequences for patients with chronic bronchitis and bronchiectasis. We hypothesised that gradations of sputum colour would correlate directly with the myeloperoxidase content of sputum and with various other indicators of the activity and consequences of bronchial diseases. METHODS: To test this hypothesis, we quantified sputum colour by reference to a sensitive nine point colour chart and correlated this assessment with indices of a number of inflammatory mediators in sputum. RESULTS: The results indicate that standardised visual measurements of sputum colour correlated strongly with myeloperoxidase, interleukin 8, leucocyte elastase (both activity and total quantity), sputum volume, protein leak, and secretory leucocyte proteinase inhibitor (p<0.001 for all). In addition, there was a strong direct correlation between leucocyte elastase and both myeloperoxidase (p<0.003) and sputum volume (p<0.001), but a strong negative correlation with secretory leucocyte proteinase inhibitor (p<0.001). CONCLUSIONS: These results indicate that sputum colour graded visually relates to the activity of the underlying markers of bronchial inflammation. The results of this simple visual analysis of sputum provides guidance concerning underlying inflammation and its damaging potential. It also provides a useful scientific tool for improving the monitoring of chronic airways diseases and response to treatment.

Relationship between airway inflammation and the frequency of exacerbations in patients with smoking related COPD.

BACKGROUND: Patients with more frequent exacerbations of chronic obstructive pulmonary disease (COPD) may have increased bronchial inflammation. Airway inflammation was measured in patients who had been thoroughly investigated with full pulmonary function testing, thoracic HRCT scanning, and sputum microbiology to examine further the relationship between exacerbation frequency and bronchial inflammation. METHODS: Airway inflammation (spontaneous sputum sol phase myeloperoxidase (MPO), elastase, leukotriene (LT)B(4), interleukin (IL)-8, secretory leukoprotenase inhibitor (SLPI), protein leakage) and serum levels of C reactive protein (CRP) were compared in 40 patients with stable, smoking related COPD, divided into those with frequent (> or =3/year) or infrequent (< or =2/year) exacerbations according to the number of primary care consultations during the preceding year. The comparisons were repeated after excluding eight otherwise clinically indistinguishable patients who had tubular bronchiectasis on the HRCT scan. RESULTS: Patients with frequent (n=12) and infrequent (n=28) exacerbations were indistinguishable in terms of their clinical, pulmonary function, and sputum characteristics, CRP concentrations, and all of their bronchial inflammatory parameters (p>0.05). The patients without evidence of tubular bronchiectasis (n=32) were equally well matched but the sputum concentrations of SLPI were significantly lower in the frequent exacerbators (n=8) in this subset analysis (p<0.05). CONCLUSIONS: There are several clinical features that directly influence bronchial inflammation in COPD. When these were carefully controlled for, patients with more frequent reported exacerbations had lower sputum concentrations of SLPI. This important antiproteinase is also known to possess antibacterial and antiviral activity. Further studies are required into the nature of recurrent exacerbations and, in particular, the regulation and role of SLPI in affected individuals.

Inflammatory mediators in spontaneously produced sputum.

Airway inflammation is currently the subject of intense research interest concerning both the nature of the inflammatory cells, proteins, and cytokines present. These data are being used to define and assess the severity, cause, prognosis, and response to treatment of airway inflammatory disease. Spontaneous sputum is a useful method for studying inflammation in the larger airways, in diseases such as asthma, bronchitis, and bronchiectasis.

Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure.

There is little known about the sequences that mediate the initiation of transcription in Bacteroides fragilis, thus transcriptional start sites for 13 new genes were determined and a total of 23 promoter regions upstream of the start sites were aligned and similarities were noted. A region at about -7 contained a consensus sequence of TAnnTTTG and upstream in the region centered at about -33, another TTTG motif was found in the majority of promoters examined. Canonical, Escherichia coli, -10 and -35 consensus sequences were not readily apparent. Mutations within the -7 motif indicated the TTTG residues were essential since changes in this sequence reduced the promoter activity to that of a no promoter control in a chloramphenicol acetyl transferase transcriptional fusion model system. Additional fusion studies indicated that the -33 region was also necessary for full activity.

Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis.

PURPOSE: Viable bacteria are often isolated from airway secretions in clinically stable patients with chronic bronchitis. We hypothesized that the number of organisms and bacterial species might be important modulators of airway inflammation. SUBJECTS AND METHODS: We performed quantitative sputum cultures in 160 stable patients [55 with chronic obstructive pulmonary disease (COPD) and normal serum alpha(1)-antitrypsin levels, 62 with COPD and severe alpha(1)-antitrypsin deficiency (PiZ), and 43 with idiopathic bronchiectasis]. The results were related to several indicators of the mechanisms and severity of airway inflammation. RESULTS: Airway bacterial load correlated with sputum myeloperoxidase level, an indirect measure of neutrophil activation and number (r = 0.50, P<0. 001); sputum neutrophil chemoattractants [interleukin-8 level (r = 0. 68, P<0.001) and leukotriene B4 level (r = 0.53, P<0.001)]; sputum leukocyte elastase activity (r = 0.55, P<0.001); and albumin leakage from serum to sputum (r = 0.26, P<0.01). Markers of inflammation increased at bacterial loads of 10(6) to 10(7) colony-forming units per milliliter, and increased progressively with increasing bacterial load. For example, the median (interquartile range) sputum myeloperoxidase level was 0.3 U/mL (0.1 to 0.5 U/mL) for patients who were not colonized or who had mixed normal oropharyngeal flora alone; 0.5 U/mL (0.2 to 0.7 U/mL) for patients with 10(5) to 10(6) colony-forming units per milliliter (P = 0.07); 0.5 U/mL (0.3 to 1.2 U/mL) for patients with 10(6) to 10(7) colony-forming units per milliliter (P<0.01); 0.7 U/mL (0.3 to 1.2 U/mL) for patients with 10(7) to 10(8) colony-forming units per milliliter (P <0.005); and 2.4 U/mL (0.7 to 4.8 U/mL) for patients with 10(8) or greater colony-forming units per milliliter (P<0.0001). The bacterial species influenced airway inflammation; for example, sputum myeloperoxidase activity was greater (P<0.005) in patients colonized with Pseudomonas aeruginosa [median 32 U/mL (interquartile range, 20 to 65 U/mL)] than those colonized with nontypeable Hemophilus influenzae [4 U/mL (2 to 31 U/mL)], which in turn was greater (P = 0.01) than among those colonized with Moraxella catarrhalis [1.1 U/mL (0.6 to 1.8 U/mL)]. We did not find a relation between bacterial load and lung function. CONCLUSIONS: The bacterial load and species contribute to airway inflammation in patients with stable chronic bronchitis. Further studies are required to determine the consequences of bacterial colonization on patient morbidity and decline in lung function.

Airways inflammation in chronic bronchitis: the effects of smoking and alpha1-antitrypsin deficiency.

Airways inflammation in chronic bronchitis is thought predominantly to be a direct consequence of neutrophil recruitment and release of elastase in response to factors such as cigarette smoke. The aims of this study were to assess the role of smoking and determine whether the serum elastase inhibitor alpha1-antitrypsin (alpha1AT) influenced the process. Airways inflammation was compared between patients with chronic obstructive bronchitis with (n=39) and without (n=42) severe alpha1AT deficiency. The authors assessed the sputum concentration of the neutrophil chemoattractants interleukin-8 (IL-8) and leukotriene (LT)B4, myeloperoxidase (MPO) as a marker of neutrophil influx, neutrophil elastase activity and its natural inhibitors, alpha1AT and secretory leukoprotease inhibitor (SLPI). Finally serum alpha1AT was measured to determine the degree of protein leakage (sputum sol serum alpha1AT ratio). Compared to current smokers, the exsmokers had a lower concentration of the chemoattractant IL-8 (p<0.05) and a lower MPO concentration, although this failed to reach conventional statistical significance (p=0.06). Patients with alpha1AT deficiency had greater inflammation in the larger airways with increased LTB4 (p<0.005), MPO (p<0.001), neutrophil elastase activity (p<0.01), protein leak (p<0.001), and were found to have a lower anti-proteinase screen with both reduced sputum alpha1AT (p<0.001) and SLPI concentrations (p<0.05). The reduction in sputum interleukin-8 levels in exsmokers may decrease neutrophil influx and thus explain the slower rate of neutrophil mediated progression of lung disease compared to subjects who continue to smoke. Patients with alpha1-antitrypsin deficiency had greater inflammation suggesting that alpha1-antitrypsin plays an important role in protecting the larger airways from the inflammatory effects of elastase activity and may explain their more rapid progression of disease.

Validation of assays for inflammatory mediators in sputum.

Commercially available immune assays are being used with increasing frequency in the study of lung inflammation. However, their performance in complex biological fluids is rarely assessed. The authors wished to assess their reliability to determine whether the results obtained in sputum samples can be easily interpreted. The reproducibility of several such assays was therefore determined together with their ability to recover known amounts of pure reagent. Sputum sol phase was obtained from several patients with chronic lung disease and used together with the reagents in a series of "spiking" and dilutional experiments. Results confirmed that the enzyme assay for myeloperoxidase and the immune assays for interleukin-8, leukotriene B4 and secretory leukoproteinase inhibitor were all reproducible (intra-assay coefficient of variation 3.8-7.7%). Furthermore, each of these assays gave >85% recovery of a "spike" with pure reagent. However, the immune assay for myeloperoxidase (although reproducible) gave poor recovery and was dependent on the degree of sample dilution and elastase content. These studies confirm that the reliabilities of fluid phase measurements should be assessed before being widely applied to complex biological samples.

Bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene B4.

Neutrophils recruited to the airways in chronic obstructive pulmonary disease (COPD) are thought to mediate tissue destruction. Neutrophil recruitment is increased during bacterial exacerbations. The inflammatory process was studied in patients with an acute exacerbation of COPD in order to ascertain the role of leukotriene B4 (LTB4). The sputum of eight subjects with a bacterial exacerbation of COPD was analysed for neutrophil products (myeloperoxidase, elastase) and chemoattractants (interleukin-8 (IL-8) and LTB4). The contribution of LTB4 to the chemotactic activity of the sputum sol phase was determined using the LTB4 receptor antagonist LY293111. The concentrations of the serum acute phase proteins alpha1-proteinase inhibitor, alpha1-antichymotrypsin and C-reactive protein were measured. All patients received appropriate broad-spectrum antibiotic treatment for 7-14 days. Initially, the sputum myeloperoxidase activity was high, indicating neutrophil influx; this was associated with high levels of IL-8 and LTB4. All these concentrations fell with treatment (p<0.01). The chemotactic activity of the sputum was raised on presentation and fell with treatment (p<0.01). LTB4 contributed approximately 30% of the total chemotactic activity on presentation; this diminished with therapy. All acute phase proteins were raised on presentation and fell with therapy (p<0.01). These findings suggest that leukotriene B4 contributes to neutrophil influx into the airway in chronic obstructive pulmonary disease and may influence disease progression.

Phenotypic changes in neutrophils related to anti-inflammatory therapy.

Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.

Evidence for excessive bronchial inflammation during an acute exacerbation of chronic obstructive pulmonary disease in patients with alpha(1)-antitrypsin deficiency (PiZ).

Patients with homozygous (PiZ) alpha(1)-antitrypsin (AAT) deficiency have not only low baseline serum AAT levels (approximately 10 to 15% normal) but also an attenuated acute phase response. They are susceptible to the development of premature emphysema but may also be particularly susceptible to lung damage during bacterial exacerbations when there will be a significant neutrophil influx. The purposes of the present study were to assess the inflammatory nature of acute bacterial exacerbations of chronic obstructive pulmonary disease (COPD) in subjects with AAT deficiency, to compare this with COPD patients without deficiency, and to monitor the inflammatory process and its resolution following appropriate antibacterial therapy. At the start of the exacerbation, patients with AAT deficiency had lower sputum AAT (p < 0.001) and secretory leukoprotease inhibitor (SLPI; p = 0.02) with higher elastase activity (p = 0.02) compared with COPD patients without deficiency. Both groups had a comparable acute phase response as assessed by C-reactive protein (CRP) but the AAT-deficient patients had a minimal rise in serum AAT (to < 6 microM). After treatment with antibiotics, in patients with AAT deficiency, there were significant changes in many sputum proteins including a rise in SLPI levels, and a reduction in myeloperoxidase (MPO) and elastase activity (p < 0. 005 for all measures); the sputum chemoattractants interleukin-8 (IL-8) and leukotriene B(4) (LTB(4)) fell (p < 0.01), and protein leak (sputum/serum albumin ratio) became lower (p < 0.01). The changes were rapid and within 3 d of the commencement of antibiotic therapy the biochemical markers had decreased significantly, but took a variable time thereafter to return to baseline values. In conclusion, patients with AAT deficiency had evidence of increased elastase activity at the start of the exacerbation when compared with nondeficient COPD patients which probably reflects a deficient antiproteinase screen (lower sputum AAT and SLPI). The increased bronchial inflammation at presentation resolved rapidly with 14 d of antibiotic therapy.