AIM: To determine induced sputum cell counts and interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha) and leukotriene B4 (LTB4) levels as markers of neutrophilic inflammation in moderate persistent asthma, and to evaluate the response to inhaled steroid therapy. METHODS: Forty-five moderate asthmatic patients and 10 non-smoker controls were included in this study. All patients received inhaled corticosteroid (800 microg of budesonide) for 12 weeks. Before and after treatment pulmonary function tests were performed, and symptom scores were determined. Blood was drawn for analysis of serum inflammatory markers, and sputum was induced. RESULTS: Induced sputum cell counts and inflammatory markers were significantly higher in patients with asthma than in the control group. The induced sputum eosinophil counts of 12 patients (26%) were found to be less than 5%, the non-eosinophilic group, and sputum neutrophil counts, IL-8 and TNF-alpha levels were significantly higher than the eosinophilic group (neutrophil, 50+/-14% versus 19+/-10%, p<0.01). In both groups, there was a significant decrease in sputum total cell counts and serum and sputum IL-8, TNF-alpha and LTB4 levels after the treatment. There was no change in sputum neutrophil counts. Although the sputum eosinophil count decreased only in the eosinophilic subjects, there was no significant difference in inflammatory markers between the groups. The symptom scores were significantly improved after treatment, while the improvement did not reach statistical significance on pulmonary function test parameters. CONCLUSION: Notably, in chronic asthma there is a subgroup of patients whose predominant inflammatory cells are not eosinophils. Sputum neutrophil counts and neutrophilic inflammatory markers are significantly higher in these patients. In the non-eosinophilic group, inhaled steroid caused an important decrease in inflammatory markers; however, there was no change in the sputum eosinophil and neutrophil counts.
Phenotyping asthma according to airway inflammation allows identification of responders to targeted therapy. Induced sputum is technically demanding. We aimed to identify predictors of sputum inflammatory phenotypes according to easily available clinical characteristics.
This retrospective study was conducted in 508 asthmatics with successful sputum induction recruited from the University Asthma Clinic of Liege. Receiver-operating characteristic (ROC) curve and multiple logistic regression analysis were used to assess the relationship between sputum eosinophil or neutrophil count and a set of covariates. Equations predicting sputum eosinophils and neutrophils were then validated in an independent group of asthmatics.
Eosinophilic (≥3%) and neutrophilic (≥76%) airway inflammation were observed in 46% and 18% of patients respectively. Predictors of sputum eosinophilia ≥3% were high blood eosinophils, FENO and IgE level and low FEV1/FVC. The derived equation was validated with a Cohen’s kappa coefficient of 0.59 (p < 0.0001). ROC curves showed a cut-off value of 220/mm3 (AUC = 0.79, p < 0.0001) or 3% (AUC = 0.81, p < 0.0001) for blood eosinophils to identify sputum eosinophilia ≥3%. Independent predictors of sputum neutrophilia were advanced age and high FRC but not blood neutrophil count.
Eosinophilic and paucigranulocytic asthma are the dominant inflammatory phenotypes. Blood eosinophils provide a practical alternative to predict sputum eosinophilia but sputum neutrophil count is poorly related to blood neutrophils.
Asthma; Induced sputum; Eosinophil; Neutrophil
Airway eosinophilia is a predictor of steroid responsiveness in steroid-naïve asthma. However, the relationship between airway eosinophilia and the expression of FK506-binding protein 51 (FKBP51), a glucocorticoid receptor co-chaperone that plays a role in steroid insensitivity in asthma, remains unknown.
To evaluate the relationship between eosinophilic inflammation and FKBP51 expression in sputum cells in asthma.
The FKBP51 mRNA levels in sputum cells from steroid-naïve patients with asthma (n = 31) and stable asthmatic patients on inhaled corticosteroid (ICS) (n = 28) were cross-sectionally examined using real-time PCR. Associations between FKBP51 levels and clinical indices were analyzed.
In steroid-naïve patients, the FKBP51 levels were negatively correlated with eosinophil proportions in blood (r = −0.52) and sputum (r = −0.57), and exhaled nitric oxide levels (r = −0.42) (all p<0.05). No such associations were observed in patients on ICS. In steroid-naïve patients, improvement in forced expiratory volume in one second after ICS initiation was correlated with baseline eosinophil proportions in blood (r = 0.74) and sputum (r = 0.76) and negatively correlated with FKBP51 levels (r = −0.73) (all p<0.0001) (n = 20). Lastly, the FKBP51 levels were the lowest in steroid-naïve asthmatic patients, followed by mild to moderate persistent asthmatic patients on ICS, and the highest in severe persistent asthmatic patients on ICS (p<0.0001).
Lower FKBP51 expression in sputum cells may reflect eosinophilic inflammation and glucocorticoid responsiveness in steroid-naïve asthmatic patients.
The monitoring of airway inflammation has assessed in bronchial asthma directly by sputum examination, and indirectly by measurements in peripheral blood. To investigate the diagnostic value of these two methods, we compared nitric oxide (NO) metabolites, eosinophils, and eosinophil cationic protein (ECP) in sputum and blood in patients with asthma and control subjects. Sputum and serum were obtained from fifteen patients with asthma, and then were examined before anti-asthma treatment, including steroid preparations. ECP was measured by fluoroimmunoassay. NO metabolites were assayed by using modified Griess reaction. Asthmatic patients, compared with control subjects, had significantly higher level of NO metabolites, higher proportion of eosinophils, and higher levels of ECP in sputum. Asthmatic patients, compared with control subjects, however, had significantly higher number of eosinophils, and were at higher levels of ECP in blood. FEV1, FEV1/FVC was negatively correlated with sputum eosinophils. The area under receiver operating characteristic (ROC) curve showed that eosinophils in sputum are significantly accurate markers than NO metabolites in sputum and blood. These findings suggest that the proportion of eosinophils in sputum have more accurate diagnostic marker of asthmatic airway inflammation than NO metabolites in sputum in differentiating asthmatic patients from control subjects.
hyperresponsiveness, induced sputum eosinophils, and exhaled nitric
oxide (NO) levels have all been proposed as non-invasive markers for
monitoring airway inflammation in patients with asthma. The aim of this
study was to compare the changes in each of these markers following
treatment with inhaled glucocorticosteroids in a single study.
randomised, double blind, placebo controlled, parallel study 25 patients with mild asthma (19-34 years, forced expiratory volume in
one second (FEV1) >75% predicted, concentration of
histamine provoking a fall in FEV1 of 20% or more
(PC20) <4 mg/ml) inhaled fluticasone propionate
(500 µg twice daily) for four weeks. PC20 to histamine,
sputum eosinophil numbers, and exhaled NO levels were determined at
weeks 0, 2, and 4, and two weeks after completing treatment. Sputum was
induced by inhalation of hypertonic (4.5%) saline and eosinophil
counts were expressed as percentage non-squamous cells. Exhaled NO
levels (ppb) were measured by chemiluminescence.
steroid treated group there was a significant increase in
PC20, decrease in sputum eosinophils, and decrease in
exhaled NO levels compared with baseline at weeks 2 and 4 of treatment. Subsequently, each of these variables showed significant worsening during the two week washout period compared with week 4. These changes
were significantly different from those in the placebo group, except
for the changes in sputum eosinophils and exhaled NO levels during the
washout period. There were no significant correlations between the
changes in the three markers in either group at any time.
of asthmatic subjects with inhaled steroids for four weeks leads to
improvements in airway hyperresponsiveness to histamine, eosinophil
counts in induced sputum, and exhaled nitric oxide levels. The results
suggest that these markers may provide different information when
monitoring anti-inflammatory treatment in asthma.
Rationale: Airway eosinophilia is typical of asthma, and many controller treatments target eosinophilic disease. Asthma is clinically heterogeneous, however, and a subgroup of people with asthma do not have airway eosinophilia. The size of this subgroup is uncertain because prior studies have not examined repeated measures of sputum cytology to determine when people with asthma have intermittent versus persistent sputum eosinophila and when they are persistently noneosinophilic.
Objectives: To determine the prevalence and clinical characteristics of the noneosinophilic asthma phenotype.
Methods: We analyzed sputum cytology data from 995 subjects with asthma enrolled in clinical trials in the Asthma Clinical Research Network where they had undergone sputum induction and measures of sputum cytology, often repeatedly, and assessment of responses to standardized asthma treatments.
Measurements and Main Results: In cross-sectional analyses, sputum eosinophilia (≥2% eosinophils) was found in only 36% of subjects with asthma not taking an inhaled corticosteroid (ICS) and 17% of ICS-treated subjects with asthma; an absence of eosinophilia was noted frequently, even in subjects with asthma whose disease was suboptimally controlled. In repeated measures analyses of people with asthma not taking an ICS, 22% of subjects had sputum eosinophilia on every occasion (persistent eosinophilia); 31% had eosinophilia on at least one occasion (intermittent eosinophilia); and 47% had no eosinophilia on every occasion (persistently noneosinophilic). Two weeks of combined antiinflammatory therapy caused significant improvements in airflow obstruction in eosinophilic asthma, but not in persistently noneosinophilic asthma. In contrast, bronchodilator responses to albuterol were similar in eosinophilic and noneosinophilic asthma.
Conclusions: Approximately half of patients with mild-to-moderate asthma have persistently noneosinophilic disease, a disease phenotype that responds poorly to currently available antiinflammatory therapy.
asthma; eosinophil; noneosinophilic; obesity; neutrophil
BACKGROUND: Airway inflammation is considered to be important in asthma but is relatively inaccessible to study. Less invasive methods of obtaining sputum from patients unable to produce it spontaneously should provide a useful investigational tool in asthma. METHODS: A method to induce sputum with inhaled hypertonic saline was modified for use in 17 asthmatic patients and 17 normal subjects who could not produce sputum spontaneously. The success rate and safety of the method, the reproducibility of cell counts, and differences in cell counts between the asthmatic and normal groups were examined. Hypertonic saline solution 3-5% was inhaled for up to 30 minutes after inhalation of salbutamol. Subjects were asked to expectorate sputum every five minutes. The quality of the sample was scored on the volume of plugs and the extent of salivary contamination. Plugs from the lower respiratory tract were selected for a total cell count and for differential cell counts of eosinophils and metachromatic cells (mast cells and basophils) in direct smears. RESULTS: Adequate samples from the lower respiratory tract were obtained in 76% of first attempts. The mean fall in the forced expiratory volume in one second (FEV1) during inhalation of saline was 5.3% and the maximum fall 20%. Eosinophil and metachromatic cell counts were reproducible (reliability coefficient 0.8 and 0.7 respectively). When compared with sputum from normal subjects sputum from asthmatic patients contained a significantly higher proportion of eosinophils (mean 18.5% (SE 3.8%) v 1.9% (0.6%)) and metachromatic cells (0.50% (0.18%) v 0.039% (0.014%)). In the asthmatic group the differential eosinophil count correlated with the baseline FEV1. CONCLUSION: Induced sputum is capable of detecting differences in cell counts between normal and asthmatic subjects and merits further development as a potential means of assessing airway inflammation in asthma.
Noneosinophilic asthma is common across asthma severities. However, in patients with moderate-to-severe disease, the absence of sputum eosinophilia cannot distinguish between asthmatic subjects with eosinophilic inflammation controlled by corticosteroids versus those in whom eosinophilic inflammation is not a component of the disease.
We sought to develop a method to quantify eosinophil proteins in airway macrophages as a novel biomarker of eosinophilic airway inflammation.
Eosinophil proteins in airway macrophages were assessed by means of flow cytometry, immunofluorescence, and cytoplasmic hue change after ingestion of apoptotic eosinophils. Airway macrophage median percentage of red-hued area in stained sputum cytospin preparations was assessed by means of image analysis from (1) subjects with mild-to-severe asthma, subjects with nonasthmatic eosinophilic bronchitis, and healthy control subjects; (2) subjects with eosinophilic severe asthma after treatment with prednisolone; and (3) subject with noneosinophilic asthma before corticosteroid withdrawal.
Eosinophil proteins were detected in airway macrophages, and cytoplasmic red hue increased after ingestion of apoptotic eosinophils. Airway macrophage percentage red-hued area was increased in subjects with moderate-to-severe asthma compared with that seen in subjects with mild asthma and healthy control subjects, was similar in those with or without a sputum eosinophilia, and was increased after corticosteroid therapy. In asthmatic subjects without sputum eosinophilia, the airway macrophage percentage red-hued area was increased in subjects who did versus those who did not have sputum eosinophilia after corticosteroid withdrawal.
Eosinophil proteins can be reliably measured in airway macrophages. In combination with sputum eosinophilia, the macrophage eosinophil protein content might further define the asthma phenotype and provide an additional tool to direct therapy.
Asthma; macrophage; eosinophil; computer-assisted image analysis; induced sputum
Asthma represents a profound worldwide public health problem. The most effective anti-asthmatic drugs currently available include inhaled β2-agonists and glucocorticoids and control asthma in about 90-95% of patients. The current asthma therapies are not cures and symptoms return soon after treatment is stopped even after long term therapy. Although glucocorticoids are highly effective in controlling the inflammatory process in asthma, they appear to have little effect on the lower airway remodelling processes that appear to play a role in the pathophysiology of asthma at currently prescribed doses. The development of novel drugs may allow resolution of these changes. In addition, severe glucocorticoid-dependent and resistant asthma presents a great clinical burden and reducing the side-effects of glucocorticoids using novel steroid-sparing agents is needed. Furthermore, the mechanisms involved in the persistence of inflammation are poorly understood and the reasons why some patients have severe life threatening asthma and others have very mild disease are still unknown. Drug development for asthma has been directed at improving currently available drugs and findings new compounds that usually target the Th2-driven airway inflammatory response. Considering the apparently central role of T lymphocytes in the pathogenesis of asthma, drugs targeting disease-inducing Th2 cells are promising therapeutic strategies. However, although animal models of asthma suggest that this is feasible, the translation of these types of studies for the treatment of human asthma remains poor due to the limitations of the models currently used. The myriad of new compounds that are in development directed to modulate Th2 cells recruitment and/or activation will clarify in the near future the relative importance of these cells and their mediators in the complex interactions with the other pro-inflammatory/anti-inflammatory cells and mediators responsible of the different asthmatic phenotypes. Some of these new Th2-oriented strategies may in the future not only control symptoms and modify the natural course of asthma, but also potentially prevent or cure the disease.
There is increasing evidence that the disorder of eosinophil apoptosis contributes to the mechanism of prominent airway inflammation in asthma. However the relationship between dysregulation of eosinophil apoptosis and severity of childhood asthma is still unclear.
Investigate the relationship between eosinophil apoptosis in induced sputum and severity of asthma in children.
Eighty-six children aged 6 to 12 years with asthma and 32 age-matched healthy controls were observed. Diagnosis of asthma was made using a clinical questionnaire, physical examination and skin prick tests (SPTs). Lung function, and induced sputum analysis were measured in all patients. Total and antigen specific IgE levels were assessesed by ELISA. Eosonophils apoptosis was determined by staining nuclei with propidium iodide, and analyzed with a FACScan. Expression Apo-1/Fas antigen (CD95) in sputum eosinophils was assessed by immunohistochemical staining techniques.
Diagnosis of asthma was confirmed by positive SPT and increased total and specific IgE levels. Asthma severity (assessed by FEV1, peak expiratory flow (PEF) variability and daily symptom scores) complied with mild and moderate asthma. The percentage sputum eosinophils was expressively increased (threshold of <3%) in all asthmatic children (compared to control group) and directly correlated with peripheral blood eosinophilia, skin sensitization, increased level of total and specific IgE and clinical symptoms of asthma and all of these markers were more significant in children with moderate asthma (P < 0.05). Asthma children showed decreased eosinophils apoptosis (“apoptotic ratio”-AR) in induced sputum as compared to controls (P < 0.001), which directly correlated with predicted value of FEV1, PEF variability and inversely with symptoms score (P = 0.005), and was significantly lower in patients with moderate asthma than those in patients with mild (P = 0.001). More of that, these parameters also correlated with decreased expression of Apo-1/Fas antigen (CD95), especially in moderate asthmatic children (P < 0.05).
Our investigation: 1. Confirms that reduced eosinophil apoptosis in induced sputum associated with increased clinical severity of asthma in children. 2. Provides additional evidence that eosinophil apoptosis may be important in the resolution of eosinophilic airway inflammation in asthma, because of their prolonged survival that maintains inflammation.
The diagnosis of childhood asthma covers a broad spectrum of pathological mechanisms that can lead to similarly presenting clinical symptoms, but may nonetheless require different treatment approaches. Distinct underlying inflammatory patterns are thought to influence responsiveness to standard asthma medication.
The purpose of the PACMAN2 study is to identify inflammatory phenotypes that can discriminate uncontrolled childhood asthma from controlled childhood asthma by measures in peripheral blood and exhaled air. PACMAN2 is a nested, case–control follow-up study to the ongoing pharmacy-based “Pharmacogenetics of Asthma medication in Children: Medication with Anti-inflammatory effects” (PACMAN) study. The original PACMAN cohort consists of children aged 4–12 years with reported use of asthma medication. The PACMAN2 study will be conducted within the larger PACMAN cohort, and will focus on detailed phenotyping of a subset of the PACMAN children. The selected participants will be invited to a follow-up visit in a clinical setting at least six months after their baseline visit based on their adherence to usage of inhaled corticosteroids, their asthma symptoms in the past year, and their age (≥ 8 years). During the follow-up visit, current and long-term asthma symptoms, medication use, environmental factors, medication adherence and levels of exhaled nitric oxide will be reassessed. The following measures will also be examined: pulmonary function, exhaled volatile organic compounds, as well as inflammatory markers in peripheral blood and blood plasma. Comparative analysis and cluster-analyses will be used to identify markers that differentiate children with uncontrolled asthma despite their use of inhaled corticosteroids (ICS) (cases) from children whose asthma is controlled by the use of ICS (controls).
Asthmatic children with distinct inflammatory phenotypes may respond differently to anti-inflammatory therapy. Therefore, by identifying inflammatory phenotypes in children with the PACMAN2 study, we may greatly impact future personalised treatment strategies, uncover new leads for therapeutic targets and improve the design of future clinical studies in the assessment of the efficacy of novel therapeutics.
Asthma; Child; Phenotypes; Inflammation; Proteomics; Volatile organic compounds; Corticosteroids
Bronchial asthma is usually associated with high sputum eosinophil levels. However, recent reports have suggested the importance of noneosinophilic asthma (NEA) as a distinct phenotype of asthma.
The aim of this study was to evaluate clinical significance of sputum eosinophilia and long-term treatment outcomes related to sputum eosinophilia in Korean asthmatics.
A total of 201 steroid-naive asthmatics who had undergone induced sputum analysis at baseline were selected from the Cohort for Reality and Evolution of Adult Asthma study population. Clinical evaluation, spirometry, a skin-prick test, a methacholine bronchial provocation test, and sputum eosinophil analysis were performed initially, and patients received the treatment recommended by the Global Initiative for Asthma. Lung function was evaluated every 6 months, and 53 patients completed 24 months of regular follow-up visits. Sputum eosinophilia was defined as a sputum eosinophil count of >3%.
Of the 201 steroid-naive asthmatics, 97 patients had NEA and 104 had eosinophilic asthma (EA). Only 52% of steroid-naive asthmatic subjects had elevated baseline sputum eosinophil levels. A higher percentage of sputum eosinophils was associated with a lower PC20 (r = -0.193; p = 0.009, Spearman correlation), but not with forced expiratory volume in one second (FEV1) (r = 0.045; p = 0.525). During the 24-month study period, the percentage change of FEV1 was significantly lower in the NEA group than in the EA group at 6, 12, 18, and 24 months (p < 0.05). The NEA group, unlike the EA group, showed no significant improvement in FEV1 at 6, 12, 18, or 24 months (p > 0.05).
A higher sputum eosinophil percentage was correlated with a higher airway hyperresponsiveness. Compared with EA patients, NEA patients had poor treatment outcomes in the 2-year follow-up of a Korean asthma cohort population.
Asthma; Induced sputum; Eosinophil; Noneosinophilic asthma
desirable to prescribe the minimal effective dose of inhaled steroids
to control asthma. To ensure that inflammation is suppressed whilst
using the lowest possible dose, a sensitive and specific method for
assessing airway inflammation is needed.
of exhaled nitric oxide (NO), sputum eosinophils, and methacholine
airway responsiveness (PC20) for monitoring airway
inflammatory changes following four weeks of treatment with an inhaled
corticosteroid (budesonide via Turbohaler) were compared. Mild stable
steroid naive asthmatic subjects were randomised into two double blind,
placebo controlled studies. The first was a parallel group study
involving three groups receiving either 100 µg/day budesonide (n = 8), 400 µg/day budesonide (n = 7), or a matched placebo (n = 6). The
second was a crossover study involving 10 subjects randomised to
receive 1600 µg budesonide or placebo. The groups were matched with
respect to age, PC20, baseline FEV1 (% predicted), exhaled NO, and sputum eosinophilia.
significant improvements in FEV1 following 400 µg and
1600 µg budesonide (11.3% and 6.5%, respectively, p<0.05). This
was accompanied by significant reductions in eosinophil numbers in
induced sputum (0.7 and 0.9 fold, p<0.05). However, levels of exhaled
NO were reduced following each budesonide dose while PC20
was improved only with 1600 µg budesonide. These results suggest
that exhaled NO and PC20 may not reflect the control of airway inflammation as accurately as the number of eosinophils in
sputum. There were dose dependent changes in exhaled NO, sputum eosinophils, and PC20 to inhaled budesonide but a plateau
response of exhaled NO was found at a dose of 400 µg daily.
the number of eosinophils in induced sputum may be the most accurate
guide to establish the minimum dose of inhaled steroids needed to
control inflammation. This, however, requires further studies involving
a larger number of patients.
Chronic obstructive pulmonary disease (COPD) and asthma may overlap and converge in older people (overlap syndrome). It was hypothesized that patients with overlap syndrome may have different clinical characteristics such as sputum eosinophilia, and better responsiveness to treatment with inhaled corticosteroid (ICS).
Sixty-three patients with stable COPD (forced expiratory volume in 1 second [FEV1] ≤80%) underwent pulmonary function tests, including reversibility of airflow limitation, arterial blood gas analysis, analysis of inflammatory cells in induced sputum, and chest high-resolution computed tomography. The inclusion criteria for COPD patients with asthmatic symptoms included having asthmatic symptoms such as episodic breathlessness, wheezing, cough, and chest tightness worsening at night or in the early morning (COPD with asthma group). The clinical features of COPD patients with asthmatic symptoms were compared with those of COPD patients without asthmatic symptoms (COPD without asthma group).
The increases in FEV1 in response to treatment with ICS were significantly higher in the COPD with asthma group. The peripheral eosinophil counts and sputum eosinophil counts were significantly higher. The prevalence of patients with bronchial wall thickening on chest high-resolution computed tomography was significantly higher. A significant correlation was observed between the increases in FEV1 in response to treatment with ICS and sputum eosinophil counts, and between the increases in FEV1 in response to treatment with ICS and the grade of bronchial wall thickening. Receiver operating characteristic curve analysis revealed 82.4% sensitivity and 84.8% specificity of sputum eosinophil count for detecting COPD with asthma, using 2.5% as the cutoff value.
COPD patients with asthmatic symptoms had some clinical features. ICS should be considered earlier as a potential treatment in such patients. High sputum eosinophil counts and bronchial wall thickening on chest high-resolution computed tomography might therefore be a good predictor of response to ICS.
COPD; asthma; HRCT; inhaled corticosteroid; pulmonary function
Inhaled glucocorticoids are efficient in protecting against asthma exacerbations, but methods to compare their efficacy vs systemic effects have only been attempted in larger multi-centre studies. The aim of the current study was therefore to directly compare the effects of two separate inhaled glucocorticoids, mometasone and budesonide, to compare the effects on the early and late asthmatic responses to inhaled allergen in patients with mild allergic asthma, and sputum eosinophils, and to relate the clinical positive effects to any systemic effects observed.
Twelve patients with documented early and late asthmatic responses (EAR and LAR) to inhaled allergen at a screening visit were randomized in a double-blind fashion to treatment with mometasone (200 μg × 2 or 400 μg × 2), budesonide (400 μg × 2) or placebo in a double-blind crossover fashion for a period of seven days. Challenge with the total allergen dose causing both an EAR and LAR was given on the last day of treatment taken in the morning. Lung function was assessed using FEV1, and systemic glucocorticoid activity was quantified using 24 h urinary cortisol.
Mometasone and budesonide attenuate both EAR and LAR to allergen to a similar degree. No significant dose-related effects on the lung function parameters were observed. Both treatments reduced the relative amount of sputum eosinophils (%) after allergen. At the dose of 800 μg daily, mometasone reduced 24 h urinary cortisol by approximately 35%. Both drugs were well tolerated.
Mometasone and budesonide are equieffective in reducing early and late asthmatic responses induced by inhaled allergen challenge. Mometasone 800 μg given for seven days partially affects the HPA axis.
allergen; asthma; budesonide; mometasone; inflammation
been associated with eosinophil activation, measured in serum, sputum,
bronchoalveolar lavage (BAL) fluid, and urine. A whole blood automated
method was developed to assess eosinophil and neutrophil activity in
terms of peroxidase content and cell morphology using the Bayer
haematology analyser. The method was applied to an in vitro stimulation
model when fMLP was added to whole blood and the samples were then
analysed for changes in granularity and shape. In addition, cells
stimulated with interleukin (IL)-8 were examined by electron microscopy.
sectional analysis was performed on venous blood from non-atopic,
non-asthmatic normal subjects (n = 37), mild (n= 46) and symptomatic
(n = 22) asthmatic patients on inhaled β2 agonist only,
and more severe asthmatic patients (n = 17) on inhaled and oral
corticosteroid therapy. Samples were analysed by the haematology
analyser and peroxidase leucograms gated using the WinMDI software program.
significant differences in the amount of light scatter by the
neutrophil populations in the symptomatic (p = 0.007) and severe
asthmatic (p = 0.0001) groups compared with the control group. However,
abnormalities in eosinophil populations were not observed. In vitro
activation of whole blood with fMLP caused similar changes in
neutrophil light scatter, suggesting that neutrophil activation is
present in peripheral blood of symptomatic asthmatic patients. IL-8
caused a change in shape of the neutrophils seen using transmission
of neutrophil activation can be seen in whole blood from patients with
asthma using a novel automated method. This may potentially be applied
to other inflammatory diseases.
Sputum cell counts have identified inflammatory subtypes of bronchitis in relatively small numbers of subjects with asthma, chronic obstructive pulmonary disease (COPD) and chronic cough in research studies. The prevalence of different subtypes of bronchitis in routine clinical practice, however, has not been reported.
To examine the heterogeneity of bronchitis and its relationship to the severity of airflow obstruction.
A retrospective cross-sectional survey based on a computerized database of spontaneous or induced sputum cell counts examined in a large university tertiary respiratory outpatient clinic.
The database contained 4232 consecutive sputum records from 2443 patients with chronic cough (39%), asthma (37%), asthma with COPD (9%), COPD (13%) and bronchiectasis (3%). Total and differential cell counts were obtained from 86% of successful sputum samples. Induced sputum provided more viable samples than spontaneous expectorate. Approximately one-third of patients with asthma and one-fifth of patients with COPD experience eosinophilic bronchitis. Asthmatic patients with moderate to severe airflow obstruction had a greater number of sputum eosinophils. There was a significantly higher number of total cell counts and percentage of neutrophils in the sputum of COPD patients with moderate and severe airflow obstruction than in those with mild airflow obstruction.
There is heterogeneity in the cellularity of sputum in various airway diseases. Patients with clinically stable airway diseases may have high sputum cell counts. During exacerbations, more patients may experience neutrophilic bronchitis. Severity of airflow obstruction is associated with eosinophilic bronchitis in patients with asthma, and neutrophilic bronchitis in patients with nonasthmatic COPD.
Asthma; Bronchitis heterogeneity; Clinical practice; COPD; Sputum cell counts
Asthma is one of the most common chronic diseases all over the world, resulting from a state of persistent sub-acute inflammation of the airways. Beside local inflammation, systemic inflammation is also present, which can be shown by increased levels of C-reactive protein (CRP). One of the most important cells in this disease is eosinophil, and sputum eosinophilia is used for its diagnosis.
The purpose of the present study was to compare and evaluate the correlation between CRP level and sputum eosinophilia in asthmatic and control subjects.
Materials and Methods:
A total of 61 patients suffering from mild-to-moderate asthma participated in this study. They were divided into two groups based on whether they used inhaled steroid or not. Sputum was induced by ultrasonic nebulizer, and then samples of peripheral venous blood were collected to measure peripheral cell count and CRP by Enzyme-linked immunosorbent assay (ELISA). Thirty-seven healthy subjects were selected and their blood samples were taken. Thirty-seven healthy subjects were selected and their blood samples were taken.
Thirty asthmatic patients in user group (14 females/16 males) with the mean age of 39.4±9.37 years, 31 asthmatic patients in non-user group (13 females/18 males) with the mean age of 35.5±8.87 years, and 37 healthy controls (17 females/20 males) were included in our study. The mean serum concentration of CRP was 2.6 μg/mL, 3.32 μg/mL, and 1.16 μg/mL in user, non-user, and control groups, respectively. Compared to healthy controls, serum concentrations of high sensitivity-CRP (hs-CRP) significantly increased in the non-user group (P=0.0001), and user group as well. (P=0.016). The number of sputum eosinophils and peripheral blood eosinophils significantly increased in the non-users compared to the healthy controls (P=0.0001, P=0.003, respectively). In the non-user group, serum hs-CRP levels correlated negatively with FEV1 and positively with numbers of sputum eosinophils, which was not statistically significant. Atopy status, age, and sex did not affect hs-CRP levels in both asthmatic groups.
It was found that serum concentrations of hs-CRP significantly increased in asthmatic patients, and in the steroid-native group it partly correlated with FEV1 and numbers of sputum eosinophils. It suggests that serum hs-CRP can indirectly indicate the degree of airway inflammation.
Asthma; C-reactive protein; eosinophilia; forced expiratory volume in one second; sputum
Patients with severe asthma have increased granulocytes in their sputum compared to patients with mild to moderate asthma.
We hypothesized that inflammatory granulocytes in sputum may identify specific asthma severity phenotypes and are associated with different patterns of inflammatory proteins in sputum supernatants.
This hypothesis was tested in 242 asthmatics enrolled in the Severe Asthma Research Program who provided sputum samples for cell count, differential cell determinations, cell lysates for Western blot, and supernatant analyses by inflammatory protein microarrays and ELISAs. ANOVA and multiple linear regression models tested mediator associations.
Stratified by sputum granulocytes, < or ≥2%eosinophils and < or ≥40%neutrophils, subjects with both increased eosinophils and neutrophils had the lowest lung function, increased symptoms and healthcare utilization. Subjects with elevated eosinophils with or without increased neutrophils had significantly increased FeNO, serum eosinophils and greater frequency of daily β-agonist use. Microarray data, stratified by granulocytes revealed 25–28 inflammatory proteins increased >2-fold in sputa with ≥40% neutrophils. Microarray analyses stratified by severity of asthma, identified 6–9 proteins increased >2-fold in sputa in subjects with severe asthma compared to nonsevere asthma. ELISA data, stratified by sputum granulocytes, showed significant increases in BDNF, IL-1β, and MIP-3α/CCL20 for those with ≥40%neutrophils; these mediators demonstrated positive associations with neutrophil counts.
Combined increased sputum eosinophils and neutrophils identified asthmatics with the lowest lung function and worse asthma control, increased symptoms and healthcare requirements. Inflammatory protein analyses of sputum supernatants found novel mediators increased in asthmatics, predominantly associated with increased sputum neutrophils.
asthma phenotypes; protein microarrays; BDNF; CXCL13; TNFSF14; CCL20; CCL18
Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.
We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.
Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.
After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).
The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.
Inhaled glucocorticosteroids are currently the most effective anti-inflammatory controller medications for treating persistent asthma. The efficacies of glucocorticoids include reducing asthma symptoms, reducing exacerbation frequency, improving quality of life, improving lung function, decreasing airway hyperresponsiveness, controlling airway inflammation, and reducing mortality. However, the treatment response to glucocorticosteroids in asthmatics varies, and certain subtypes of asthma, such as refractory asthma, respond poorly to high-dose inhaled glucocorticoid and systemic steroids. The medical costs of treating refractory asthmatics represent about 50% of the total healthcare cost for asthma. A thorough understanding of the mechanisms of glucocorticoid action, patient responses to glucocorticoids, and steroid resistance observed in refractory asthmatics is necessary for the targeted development of therapeutic drugs. This review discusses the characteristics of severe refractory asthmatics and the mechanisms of steroid response and resistance in asthma treatment.
Glucocorticoids; Drug resistance; Asthma
Molecular microbiological analysis of airway samples in asthma has demonstrated an altered microbiome in comparison to healthy controls. Such changes may have relevance to treatment-resistant severe asthma, particularly those with neutrophilic airway inflammation, as bacteria might be anticipated to activate the innate immune response, a process that is poorly steroid responsive. An understanding of the relationship between airway bacterial presence and dominance in severe asthma may help direct alternative treatment approaches.
We aimed to use a culture independent analysis strategy to describe the presence, dominance and abundance of bacterial taxa in induced sputum from treatment resistant severe asthmatics and correlate findings with clinical characteristics and airway inflammatory markers.
Induced sputum was obtained from 28 stable treatment-resistant severe asthmatics. The samples were divided for supernatant IL-8 measurement, cytospin preparation for differential cell count and Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling for bacterial community analysis.
In 17/28 patients, the dominant species within the airway bacterial community was Moraxella catarrhalis or a member of the Haemophilus or Streptococcus genera. Colonisation with these species was associated with longer asthma disease duration (mean (SD) 31.8 years (16.7) vs 15.6 years (8.0), p = 0.008), worse post-bronchodilator percent predicted FEV1 (68.0% (24.0) vs 85.5% (19.7), p = 0.025) and higher sputum neutrophil differential cell counts (median (IQR) 80% (67–83) vs 43% (29–67), p = 0.001). Total abundance of these organisms significantly and positively correlated with sputum IL-8 concentration and neutrophil count.
Airway colonisation with potentially pathogenic micro-organisms in asthma is associated with more severe airways obstruction and neutrophilic airway inflammation. This altered colonisation may have a role in the development of an asthma phenotype that responds less well to current asthma therapies.
High mobility group protein B1 (HMGB1) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of HMGB1 in plasma and sputum of patients with asthma and COPD across disease severity needs to be defined. The objective of the study was to examine the induced sputum and plasma concentrations of HMGB1 in COPD and asthmatic patients to determine differences in HMGB1 levels between these diseases and their relationship with airway obstruction and inflammatory patterns. A total of 147 participants were enrolled in this study. The participants included 34 control subjects, 61 patients with persistent asthma (according to the Global Initiative for Asthma [GINA] guidelines) and 47 patients with stable COPD (stratified by Global Initiative for Chronic Obstructive Lung Disease [GOLD] status). Spirometry was performed before sputum induction. HMGB1 levels in induced sputum and plasma were determined by enzyme-linked immunosorbent assay. Sputum and plasma concentrations of HMGB1 in patients with asthma and COPD were significantly higher than concentrations in control subjects and were significantly negatively correlated with forced expiratory volume in 1 s (FEV1), FEV1 (% predicted) in all 147 participants. The levels of HMGB1 in induced sputum of COPD patients were significantly higher than those of asthma patients and healthy controls (P < 0.001). This difference was present even after adjusting for sex, age, smoking status, daily dose of inhaled corticosteroids and disease severity. There were no significant differences in HMGB1 levels between patients with eosinophilic and noneosinophilic asthma. HMGB1 levels in asthmatic and COPD patients were positively correlated with neutrophil counts and percentage of neutrophils. In multivariate analysis, the two diseases (asthma and COPD) and disease severity were independent predictors of sputum HMGB1, but not smoking, age or use of inhaled corticosteroids. In conclusion, these data support a potential role for HMGB1 as a biomarker and diagnostic tool for the differential diagnosis of asthma and COPD. The importance of this observation on asthma and COPD mechanisms and outcomes should be further investigated in large prospective studies.
BACKGROUND--Sputum analysis provides a non-invasive method of examining the airway secretions of subjects with asthma in order to better understand the inflammatory process. Increased proportions of eosinophils are generally seen in the sputum of subjects with asthma, especially when there is an exacerbation. An unexpected observation in the sputum of subjects with mild exacerbations of asthma is reported. METHODS--Thirty four consecutive subjects with symptoms consistent with a mild exacerbation of asthma were recruited for a treatment study. Inclusion criteria required persistent symptoms of chest tightness, dyspnoea, or wheezing for two weeks (without spontaneous improvement or alteration in dose of inhaled corticosteroid) and a forced expiratory volume in one second (FEV1) that was reversible to more than 75% predicted or known best to ensure the exacerbation was mild. Sputum (spontaneous or induced with hypertonic saline) from all subjects was examined for differential cell counts. Eosinophilic sputum was defined as > or = 4% eosinophils on two occasions or > 10% eosinophils once. Clinical characteristics, sputum differential counts, and measurements of airways obstruction were compared between the subjects with and without sputum eosinophilia. RESULTS--Almost half of the subjects (16 of 34) considered to have mildly uncontrolled asthma had no sputum eosinophilia. In comparison with the subjects who had sputum eosinophilia the non-eosinophilic group had less airways obstruction (FEV1% predicted 88% v 70%) and less severe airways hyperresponsiveness (PC20 methacholine 0.45 mg/ml v 0.13 mg/ml). There was no difference between the groups in the type or prevalence of symptoms, history of recent infections, smoking, relevant allergen exposure, or use of inhaled corticosteroid. CONCLUSIONS--Symptoms of mildly uncontrolled asthma are not always associated with eosinophilic airways inflammation as measured by sputum analysis. The causes and treatment of the non-eosinophilic condition require further investigation.
Background: The relationship between the clinical pattern of asthma and airway inflammation in childhood asthma is poorly characterised, yet underpins the treatment recommendations in current asthma guidelines. A study was undertaken to examine the relationship between airway inflammation and clinical asthma in children.
Methods: Children with asthma (n=146) and healthy controls (C, n=37) were recruited from primary and specialist clinics. Sputum induction and hypertonic saline challenge were performed.
Results: As the frequency of asthma episodes in the past 12 months increased, there were significant increases in sputum eosinophils (median; infrequent episodic (IE) 1.5%, frequent episodic (FE) 2.3%, persistent (P) 3.8%, control (C) 1.0%; p=0.002), sputum eosinophil cationic protein (ECP) (IE 113 ng/ml, FE 220, P 375, C 139; p=0.003), and desquamated bronchial epithelial cells (IE 2.0%, FE 6.0%, P 5.0%, C 2.5%; p=0.04). Treatment intensity was also associated with increased sputum eosinophils (p=0.005). The relationships between other severity markers (current symptoms, lung function) were less strong.
Conclusion: Children with more frequent episodes of clinical asthma exhibit increasing airway inflammation that is characterised by sputum eosinophilia and bronchial epithelial desquamation. The results support clinical assessment by frequency of wheezing episodes over the past 12 months when determining anti-inflammatory treatment requirements, and indicate that current symptoms are determined by mechanisms in addition to sputum eosinophilia.