Methods to examine sputum for indices of airway inflammation are evolving. We have examined the repeatability and the validity of an improved method to measure sputum cells and fluid-phase eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), albumin, fibrinogen, tryptase, and interleukin-5 (IL-5). Sputum was induced with hypertonic saline twice within 6 d in 10 healthy subjects, 19 stable asthmatics, and 10 smokers with nonobstructive bronchitis. The method included the processing of freshly expectorated sputum separated from saliva, treatment with a fixed proportion of dithiothreitol 0.1% followed by Dulbecco's phosphate-buffered saline, making cytospins, and collecting the supernatant. The reproducibility of measurements, calculated by the intraclass correlation coefficient, was high for all indices measured with the exception of total cell counts and proportion of lymphocytes. Asthmatics, in comparison with healthy subjects and smokers with bronchitis, had a higher proportion of sputum eosinophils (median percent 5.2 versus 0.5 and 0.3), metachromatic cells (0.3 versus 0.07 and 0.08), ECP (1,040 micrograms/L versus 288 and 352), MBP (1,176 micrograms/L versus 304 and 160), and EDN (1,512 micrograms/L versus 448 and 272). Asthmatics differed from healthy subjects, but not from smokers with bronchitis, in the proportion of neutrophils (46.9% versus 24.1%), albumin (704 versus 288 micrograms/mL), and fibrinogen (2,080 versus 440 ng/mL). Smokers with bronchitis showed a trend for a higher neutrophil count and levels of albumin and fibrinogen than healthy subjects. The proportion of sputum eosinophils correlated positively with ECP, MBP, EDN, albumin and fibrinogen levels, and metachromatic cell counts correlated with tryptase. In asthmatics, IL-5 correlated with eosinophil counts. There was a significant negative correlation between sputum indices and expiratory flows and methacholine PC20. Thus, the methods of measuring cell and fluid phase markers in induced sputum used in this study are reproducible and valid. They can therefore be used to reliably measure these indices of airway inflammation.

Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements.

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the growing properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil-mediated mucosal diseases are conceptualized.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2222.2008.02958.x

Eosinophils: Biological Properties and Role in Health and Disease

Induced sputum cell counts provide a relatively noninvasive method to evaluate the presence, type, and degree of inflammation in the airways of the lungs. Their interpretation requires a knowledge of normal values from a healthy population. The objective was to examine the total and differential cell counts in induced sputum from a sample of healthy adults. A total of 118 healthy nonsmoking adults were studied. None had asthma or airflow obstruction (negative history, FEV(1) >/= 80% predicted, ratio of FEV(1) to vital capacity [FEV(1)/VC] >/= 80%, methacholine PC(20) >/= 16 mg/ml). Forty-six were atopic. Sputum induction produced an adequate sample in 96 subjects [53 males, mean age (range) 36 (18 to 60) yr]. The expectorate was processed within 2 h; sputum was selected, treated with dithiothreitol, filtered, and examined in a hemocytometer for total cell count and viability and on Wright-stained cytospins for a differential cell count. The mean, median (90th percentile) total cell count was 4.1, 2.4 (9.7) x 10(6) cells/g and cell viability was 69.6, 72.0 (89.7)%. The proportions of eosinophils were 0.4, 0.0 (1.1)%, neutrophils 37.5, 36.7 (64.0)%, macrophages 58.8, 60.8 (86.1)%, lymphocytes 1.0, 0.5 (2.6)%, metachromatic cells 0.0, 0.0 (0.04)%, and bronchial epithelial cells 1.6, 0.3 (4.4)%, respectively. Female gender and atopy were associated with a significant elevation of eosinophils; mean difference between male/female was 0.3% (p = 0.043) and between atopic/nonatopic 0.4% (p = 0.024). This study has identified reference values for total and differential cell counts in induced sputum of healthy adults.

https://www.atsjournals.org/doi/pdf/10.1164/ajrccm.161.2.9903097

Induced Sputum Cell Counts in Healthy Adults

Venge P. Soluble markers of allergic inflammation.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1398-9995.1994.tb00765.x

Soluble markers of allergic inflammation

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.

https://erj.ersjournals.com/content/erj/21/6/1050.full.pdf

Indirect airway challenges

It has recently been recognized that platelets can be activated independently of classical aggregation [1,2]. IgE receptor mechanisms support platelet involvement in allergic disease [3]. A pathogenetic role for platelets in asthma was initially proposed by Morley et al. [4]. Evidence was three-fold: animal models of allergic inflammation; clinical studies of platelet activation; and recognition of the potential of platelet-activating factor (PAF) as a mediator of the pathophysiology of asthma. The increasing literature regarding the inflammatory properties of PAF is outside the scope of this review. Inter-connections between the kinin, complement and clotting cascades have also been omitted. There have been long-standing suggestions that coagulation may be abnormal in the atopic or asthmatic state. In an early study antigen bronchoprovocation was associated with profound reduction in plasma recalcification time [5]. The paper by Szczeklik et al. [6] in this issue once again raises questions of platelet and coagulation abnormalities in asthma. In aspirin-induced asthma there is good evidence of specific abnormalities of platelet response to non-steroidal anti-inflammatory drugs and this is not addressed. The value of the platelet as a cellular model of this asthma syndrome has recently been reviewed [7]. The first question is whether platelets are abnormal in the atopic state or in asthmatic patients. There is surprisingly little physical information regarding platelets from asthmatics. It was suggested that mean platelet volume was 11 % greater in 11 atopic subjects compared with normal controls [8]. Szczeklik et al. [9] found a small but significant increase in platelet numbers in atopy with a small increase in platelet mass but a normal mean platelet volume. Some evidence of altered platelet biochemistry has been reported in atopy. Arachidonic acid metabolism was increased in response to thrombin and various concentrations of collagen [8]. Others reported that platelets from asthmatic patients produced an increased ratio of lipoxygenase to cyclo-oxygenase products from excess exogenous radiolabelled arachidonate in comparison with non-atopic controls [10]. Reduced serotonin uptake by platelets from patients with exacerbations of asthma has been associated with increased serum and whole blood serotonin concentrations [11]. In stable asthma, platelet serotonin uptake was normal [12]. Platelet numbers have been found to decrease in

Platelet and clotting abnormalities in asthma.

Bronchial inflammation in mild asthma has been investigated using bronchoscopical techniques The safety of bronchoscopy in patients with more severe asthma has been questioned We have used the non-invasive technique of hypertonic saline (HS) inhalation to induced sputum samples for cellular analysis whilst simultaneously yielding a measure of bronchial responsiveness Ten normal subjects and a heterogenous group of 24 asthmatic patients (range % predicted FEV1 433-1115) underwent HS challenge Sputum samples induced were analysed Total and differential cell counts between the two groups were compared The association between bronchial responsiveness to HS and sputum cell counts was examined in the asthma group Mean maximum fall in FEV1 for normal subjects was 40 (21-59, 95% CI)% after saline Geometric mean PD20HS for asthma patients was 77 (range 068-4092)ml Adequate sputum samples were obtained from 9/10 normals and 23/24 asthmatic patients Sputum from normal subjects contained a median of 38 (28-81, interquartile range)% eosinophils compared with 176 (89-341)% in sputum from asthma patients (P < 0001) Sputum from asthma patients contained fewer of all other cell types compared with normals, with the difference in macrophages reaching significance There was no correlation between PD20HS and cell count for any cell type in asthma subjects Analysis of induced sputum represents a simple, safe, non-invasive and well-tolerated method of assessment of bronchial inflammation, suitable for use in patients with a range of asthma severity There was no relationship between inflammation, as assessed by sputum cell counts and a measure of 'indirect' bronchial responsiveness

Non‐invasive assessment of bronchial inflammation in asthma: no correlation between eosinophilia of induced sputum and bronchial responsiveness to inhaled hypertonic saline

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Platelet and clotting abnormalities in asthma.

Non‐invasive assessment of bronchial inflammation in asthma: no correlation between eosinophilia of induced sputum and bronchial responsiveness to inhaled hypertonic saline