Cytokine responses of peripheral blood mononuclear cells to allergen do not identify asthma or asthma phenotypes.

BACKGROUND: Asthmatic patients are often differentiated based on their atopic status (atopic or nonatopic) and type of bronchitis (eosinophilic, neutrophilic, both, or neither). There is evidence supporting a central role for the T cell in asthma, but the role of allergen-induced T cell cytokines in driving disease in different asthma phenotypes remains unclear. OBJECTIVE: To investigate the hypothesis that peripheral blood mononuclear cells (PBMCs) from asthma patients with different phenotypes would react characteristically to a panel of common aeroallergens. METHODS: We incubated PBMCs from 41 asthma patients and 8 healthy controls with allergen and assessed PBMC proliferation by (3) H-thymidine incorporation and the production of the cytokines IL-5, IL-17A, IL-23, IL-10, and IFN-γ by ELISA. RESULTS: No differences in PBMC proliferation or cytokine production were found in patients with asthma, compared with healthy controls, or between patients with different asthma phenotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Peripheral blood mononuclear cell cytokine responses to allergen are not able to assist in the discrimination between disease state, atopic status, or type of bronchitis in asthma.

Safety and efficacy of a CXCR2 antagonist in patients with severe asthma and sputum neutrophils: a randomized, placebo-controlled clinical trial.

BACKGROUND: Increased numbers of neutrophils are reported in the airways of patients with severe asthma. It is not clear if they contribute to the lack of control and severity. There are currently no strategies to investigate this by decreasing neutrophil numbers in the airways. OBJECTIVE: To investigate the safety and efficacy of SCH527123, a selective CXCR2 receptor antagonist, in patients with severe asthma and increased number of neutrophils in sputum. METHODS: In a randomized, double-blind, parallel study, patients with severe asthma and sputum total cell count < 10 × 10(6) /g and neutrophils > 40% were randomized to SCH527123, 30 mg daily PO (n = 22) or placebo (n = 12) for 4 weeks. Primary end-points were safety and change in sputum and blood neutrophil counts. Secondary end-points were change in asthma control questionnaire (ACQ) score, minor and major exacerbations, spirometry and sputum neutrophil activation markers. RESULTS: The SCH 527123 caused a mean reduction of 36.3% in sputum neutrophil percentage compared to a 6.7% increase in the placebo arm (P = 0.03). The mean absolute neutrophil count in blood was reduced by 14% at the end of 4 weeks, but recovered by the 5th week. There were no differences in the overall rates of adverse events among the groups. There were fewer mild exacerbations (1.3 vs. 2.25, P = 0.05) and a trend towards improvement in the ACQ score (mean difference between groups of 0.42 points, P = 0.053). No statistically significant changes were observed in forced expiratory volume in 1 s (FEV (1)), sputum myeloperoxidase, IL8 or elastase. CONCLUSIONS: The SCH527123 is safe and reduces sputum neutrophils in patients with severe asthma. CLINICAL RELEVANCE: This new treatment provides an opportunity to investigate the role of neutrophils in severe asthma with potential clinical benefits. Larger studies of longer duration are needed to evaluate the impact on other outcomes of asthma including exacerbations.

Monitoring sputum eosinophils in mucosal inflammation and remodelling: a pilot study.

Normalisation of eosinophil counts in sputum of asthmatic patients reduces eosinophilic exacerbations. However, the effect of this strategy on airway remodelling remains to be determined. We compared bronchial inflammation and collagen deposition after 2 yrs of treatment guided by either sputum eosinophils (sputum strategy, SS) or by clinical criteria (clinical strategy, CS). As a pilot study, 20 mild asthmatic patients were randomly assigned to CS or SS strategies. Bronchial biopsies were obtained when minimum treatment needed to maintain control was identified and this was continued for 2 yrs. Biopsies were immunostained for inflammatory cells, mucin 5A (MUC5A) and collagen. The mean dose of inhaled corticosteroids at the start and end of the study was similar in both SS and CS groups. Forced expiratory volume in 1 s increased in both groups at the study end. In SS, mucosal lymphocyte and eosinophil counts, but not neutrophils, were reduced at the end of the study. In CS, only activated eosinophil and neutrophil counts decreased. MUC5A staining decreased in SS but not CS. No change in collagen deposition underneath the basement membrane was observed in either strategy. Treatment strategies that normalise sputum eosinophils also reduce mucosal inflammatory cells and MUC5A expression, but do not change subepithelial collagen deposition in mild to moderate asthma.

The definition and diagnosis of asthma.

The diagnosis of asthma depends on what we mean by the word. Its definition continues to be controversial because there is no single genetic or environmental cause. Addressed from a descriptive point of view, the disease components include airway inflammation, symptoms, variable airflow limitation and chronic airflow limitation. The essentialist definition conveys the message that asthma is a separate disease entity, fails to identify a primary defining characteristic which separates it from other diseases and is long winded. These disadvantages are overcome by the nominalist definition of asthma in which the word 'asthma'refers to an abnormality of airway function, specifically to wide variations in airflow limitation over short periods of time. In patients with asthma the other components of airway disease need to be considered. These have separate nominalist definitions and especially include different types of bronchitis for airway inflammation and chronic obstructive pulmonary disease for chronic airflow limitation. What is present will vary between and within patients. The accurate diagnosis of asthma and of other components of disease all require objective measurements. Currently spirometry and airway responsiveness should be available to the general practitioner, who sees milder disease, and additional quantitative sputum cell counts in specialist practice, where moderate to severe disease is more prevalent. Such measurements characterize the patient, identify heterogeneity and allow treatment to be personalized.

Effect of obesity on airway inflammation: a cross-sectional analysis of body mass index and sputum cell counts.

BACKGROUND: Several observational studies have demonstrated an association between obesity and asthma. Studies evaluating exhaled nitric oxide levels and obesity have revealed that a higher body mass index (BMI) is associated with elevated exhaled nitric oxide levels. Airway inflammation using sputum cell counts has not been assessed in obese patients with airway diseases. OBJECTIVE: The primary aim of this study was to determine whether obesity (based on BMI) is associated with eosinophilic or neutrophilic bronchitis. METHODS: The results from a database of induced sputum cell counts were compared with BMI and analysed using correlation statistics, regression and parametric and non-parametric analysis. RESULTS: Seven-hundred and twenty-seven adult participants with an equal number of sputum samples were included in the analysis. BMI varied from 14.5 to 55 kg/m(2). Sputum total cell count (mean+/-SD: 12.9 x 10(6) cell/g+/-21.5), eosinophil percent (median; min to max: 0.3%; 0-89.0), and neutrophil percent (mean+/-SD: 63.5+/-26.6%) were within normal limits. Participants with asthma had a higher percentage of sputum eosinophils than those without asthma (P=0.01). However, there was no difference in the total or differential cell counts among the obese and non-obese participants, when the data were analysed according to BMI category, gender, dose of inhaled corticosteroid, and presence or absence of asthma. CONCLUSION: In this large sample of adult asthmatic and non-asthmatic participants, there was no association between BMI and airway inflammation measured by sputum cell counts. Other mechanisms to explain the relationship between obesity and asthma will need to be explored if this association is to be better understood.

The efficiency of sputum cell counts in cystic fibrosis.

BACKGROUND: Technical factors relating to processing viscid sputum in cystic fibrosis (CF) and their influence on the reproducibility and validity of cell counts need to be evaluated. In addition, the methods need to be standardized so that they can be applied clinically and in research. OBJECTIVE: To examine the efficiency, reliability and validity of processing small volumes of spontaneously expectorated sputum from subjects with CF. METHODS: Sputum was collected from adults with CF (n=35) and compared with sputum from adults with infective bronchitis or bronchiectasis (IB/B) (n=16), or with asthma or chronic obstructive pulmonary disease (AS/COPD) (n=25). Selected sputum (100 mg to 200 mg) was processed with dithiothreitol (0.1%) and filtered. Total cell count (TCC) and viability were obtained in a counting chamber and cytospins were prepared and stained with Wright's for a differential cell count. Sputum and filter remnant were processed for TCC, viability and differential cell count, and the efficiency was determined by comparing the mean loss in cell yield to the filter. Two different portions from the same sputum sample were processed for cell counts to determine reproducibility. Results were compared with those from IB/B and AS/COPD groups. RESULTS: Efficiency of cell dispersal was excellent and similar to that in AS/COPD and IB/B groups. Reproducibility of cell counts from two portions of a sputum sample was high (>or=0.80). CF sputum demonstrated a raised TCC and neutrophilia similar to IB/B but significantly higher than AS/COPD. CONCLUSION: The selection method of evaluating cell counts in viscid CF sputum is efficient, reproducible and valid.

Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations.

One important goal of asthma treatment is to reduce exacerbations. The current authors investigated if the use of sputum cell counts to guide treatment would achieve this goal. A total of 117 adults with asthma were entered into a multicentre, randomised, parallel group-effectiveness study for two treatment strategies over a 2-yr period. In one strategy (the clinical strategy: CS) treatment was based on symptoms and spirometry. In the other (the sputum strategy: SS) sputum cell counts were used to guide corticosteroid therapy to keep eosinophils250 microg, and was due to fewer eosinophilic exacerbations. The cumulative dose of corticosteroid during the trial was similar in both groups. Monitoring sputum cell counts was found to benefit patients with moderate-to-severe asthma by reducing the number of eosinophilic exacerbations and by reducing the severity of both eosinophilic and noneosinophilic exacerbations without increasing the total corticosteroid dose. It had no influence on the frequency of noneosinophilic exacerbations, which were the most common exacerbations.

Stable COPD: predicting benefit from high-dose inhaled corticosteroid treatment.

The role of inhaled corticosteroids in the management of chronic obstructive pulmonary disease (COPD) remains controversial. The purpose of this study was to evaluate whether sputum eosinophilia (defined as eosinophils > or = 3%) predicts clinical benefit from inhaled corticosteroid treatment in patients with smoking-related clinically stable moderate-to-severe COPD. Forty consecutive patients with effort dyspnoea (mean age 67 yrs; 52 pack-yr smoking history; post-bronchodilator forced expiratory volume in one second (FEV1) <60% predicted, consistent with moderate-to-severe smoking-related chronic airflow limitation) were enrolled. Subjects were treated with inhaled placebo followed by inhaled budesonide (Pulmicort Turbuhaler 1,600 microg.day(-1)), each given for 4 weeks. While the treatment was single-blind (subject level), sputum cell counts before and after treatment interventions were double-blind, thus removing bias. Outcome variables included spirometry, quality-of-life assessment and 6-min walk test. Sputum eosinophilia was present in 38% of subjects. In these, budesonide treatment normalised the eosinophil counts and, in comparison to placebo treatment, resulted in clinically significant improvement in the dyspnoea domain of the disease-specific chronic respiratory questionnaire (0.8 versus 0.3) and a small but statistically significant improvement in post-bronchodilator spirometry (FEV1 100 mL versus 0 mL; p<0.05). In conclusion, sputum eosinophilia predicts short-term clinical benefit from high-dose inhaled corticosteroid treatment in patients with stable moderate-to-severe chronic obstructive pulmonary disease.

Steroid naive eosinophilic asthma: anti-inflammatory effects of fluticasone and montelukast.

BACKGROUND: Inhaled corticosteroids and leukotriene receptor antagonists reduce airway eosinophilia and have been used as first line anti-inflammatory therapy for mild persistent asthma. METHODS: A multicentre, randomised, placebo controlled, parallel group study was performed to compare the anti-inflammatory effects of fluticasone propionate and montelukast as measured by sputum eosinophils in 50 adults with symptomatic steroid naive asthma and sputum eosinophilia of > or =3.5%. RESULTS: Eighteen patients received low dose fluticasone (250 mug/day), 19 received montelukast (10 mg/day), and 13 were given placebo for 8 weeks. Fluticasone treatment resulted in a greater reduction in sputum eosinophils (geometric mean (SD) 11.9 (2.3)% to 1.7 (5.1)%) than montelukast (10.7 (2.3)% to 6.9 (3.8)%; p = 0.04) or placebo (15.4 (2.4)% to 7.8 (4.2)%; p = 0.002), and improvement in FEV(1) (mean (SD) 2.6 (0.9) l to 3.0 (0.9) l) than montelukast (2.8 (0.7) l to 2.8 (0.9) l; p = 0.02) or placebo (2.4 (0.8) l to 2.4 (0.9) l; p = 0.01). Treatment with fluticasone suppressed sputum eosinophilia within a week while montelukast only attenuated it. The effect of montelukast was maximal at 1 week and was maintained over 4 weeks. The effect of fluticasone was maintained over 8 weeks while that of montelukast was not. CONCLUSIONS: Montelukast is not as effective as low dose fluticasone in reducing or maintaining an anti-inflammatory effect in steroid naive eosinophilic asthma.

Failure of montelukast to reduce sputum eosinophilia in high-dose corticosteroid-dependent asthma.

Sputum eosinophilia is a sensitive predictor of benefit from corticosteroid treatment. Montelukast is a cysteinyl leukotriene antagonist, which also reduces sputum and blood eosinophils. The present study examined the possibility that montelukast has an added eosinophil-lowering effect in subjects with asthma who are corticosteroid responsive but relatively corticosteroid resistant. A total of 14 clinically stable adults with asthma requiring minimum treatment with a high-dose inhaled steroid or prednisone, with baseline sputum eosinophilia (> or =5%), were randomised to receive 4 weeks of 10 mg montelukast or placebo daily in a double-blind crossover trial. The primary outcome was the effect of treatment on the percentage of sputum eosinophils. Secondary outcomes were changes in the blood eosinophil count, symptoms, forced expiratory volume in one second, peak expiratory flow and the need for salbutamol. The median (interquartile range, i.e. 75th-25th centile) for sputum eosinophils at baseline was 15.7% (22). The effect of adding montelukast was not significantly different from that of placebo, sputum eosinophils being 9.3% (18.9) after montelukast and 11.3% (22.8) after placebo. No difference was detected on secondary outcomes. No crossover interactions were observed. In conclusion, the addition of montelukast to existing high-dose corticosteroid therapy in subjects with asthma with elevated sputum eosinophils does not provide additional attenuation of airway eosinophilia.

Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets.

Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry. Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20 C after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing. Compared to immediate processing, percentages of total lymphocytes and T-suppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used. In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects.

A method to preserve sputum for delayed examination.

Examination of sputum cell counts is limited by the need to process samples within hours of expectoration. The validity and repeatability of a method to preserve sputum for delayed processing and examination were investigated in this study. Portions of selected sputum from 39 subjects were dispersed with dithiothreitol (routine method), or were fixed in a dithiothreitol-formaldehyde mixture for 48-72 h before dispersal with trypsin (preservation method). Total and differential cell counts using the two methods were compared; within-method repeatability of the preservation method was also examined. The intraclass correlation coefficient (ICC) for total cell counts and percentage of eosinophils, neutrophils and macrophages in sputum processed by the two methods was 0.89, 0.86, 0.91 and 0.90, respectively. Within-method repeatability (ICC) of the preservation method for the same cellular indices was 0.99, 0.94, 0.97 and 0.97, respectively. The interobserver repeatability for eosinophils, neutrophils and macrophages was 0.96, 0.97 and 0.97 using the preservation method, and 0.96, 0.99 and 0.99 using the routine method, respectively. This method of sputum preservation and dispersal is valid, reliable and convenient, and may be used for delayed processing and examination.

Indirect airway challenges.

Indirect challenges act by causing the release of endogenous mediators that cause the airway smooth muscle to contract. This is in contrast to the direct challenges where agonists such as methacholine or histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Direct airway challenges have been used widely and are well standardised. They are highly sensitive, but not specific to asthma and can be used to exclude current asthma in a clinic population. Indirect bronchial stimuli, in particular exercise, hyperventilation, hypertonic aerosols, as well as adenosine, may reflect more directly the ongoing airway inflammation and are therefore more specific to identify active asthma. They are increasingly used to evaluate the prevalence of bronchial hyperresponsiveness and to assess specific problems in patients with known asthma, e.g. exercise-induced bronchoconstriction, evaluation before scuba diving. Direct bronchial responsiveness is only slowly and to a modest extent, influenced by repeated administration of inhaled steroids. Indirect challenges may reflect more closely acute changes in airway inflammation and a change in responsiveness to an indirect stimulus may be a clinically relevant marker to assess the clinical course of asthma. Moreover, some of the indirect challenges, e.g. hypertonic saline and mannitol, can be combined with the assessment of inflammatory cells by induction of sputum.

Airway inflammation in COPD: physiological outcome measures and induced sputum.

Chronic obstructive pulmonary disease (COPD) is a result of airway inflammation, and the best predictor of COPD is the early detection of airflow limitation by spirometry. The Global Initiative for Obstructive Lung Disease Workshop Report defines airflow limitation using simple spirometric indices. Available guidelines categorise the severity of COPD using forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), with symptoms playing a minor role in the assessment. Current standards define COPD by progressive loss of FEV1, and thus longitudinal decline in FEV1 will be the primary outcome variable for intervention studies aimed at preventing or reducing the loss of pulmonary function. There is evidence, however, that the variable FEV1/FVC and FEV1 are often not measured properly in all settings. This article will discuss the roles of physiological measurements in diagnosing COPD and physiological outcome measures for COPD. It does not formally compare physiological measures with other outcome measures, such as symptoms or quality of life. Additionally, improved treatment of established disease requires a better understanding of the inflammatory process and its clinical effects and treatment. The inflammatory process, and how drugs affect it, can be studied noninvasively or relatively noninvasively by using refined methods of examining spontaneous or induced sputum. Enhanced understanding of the use of induced sputum will assist in predicting patients' responses to short- and long-term inhaled corticosteroid treatment, and the methods of sputum examination need to be simplified so that they can be applied more easily to clinical practice.