Study design and participants

This was a comparative cross-sectional study to measure and compare total IgE and Der p 1-specific IgE levels in the induced sputum of asthmatics and in a group of healthy volunteers. Patients and controls were matched for age, sex, and disease severity; and asthma control for allergic and non-allergic groups. Patients were consecutively enrolled from the outpatient asthma unit of our institution, a tertiary referral university hospital in Spain, between January and December 2013.

Total and Der p 1-specific IgE were measured in both serum and induced sputum. The IgE levels in serum and sputum were compared to determine the correlation between IgE levels in these two fluids.

Definition of allergic and nonallergic asthma

We defined asthma as a history of variable respiratory symptoms and evidence of variable expiratory airflow limitation. All patients had a positive bronchodilator test or a document positive methacholine challenge test. Asthma severity was defined according to the Global Initiative for Asthma Management (GINA)[17].

Allergic asthma was defined as asthma with 1) positive skin prick test to aeroallergens and/or 2) clinically-significant Der p 1-specific IgE according to the recommendations of the Committee of Skin Tests of the European Academy of Allergy and Clinical Immunology (EAACI) international task force[18]. Patients sensitized to various allergens were included only if dust mite allergy was the only clinically relevant one; if they showed symptoms in relation to other exposures they were excluded. Nonallergic asthma was defined as asthma with: 1) negative prick test, 2) negative Der p 1-specific IgE in serum; and 3) negative Phadiatop test (ImmunoCAP immunofluoroassay; Phadia ThermoFisher Scientific)[19]. D. pteronyssinus was the selected allergen because is the perennial allergen more prevalent in our geographic area.

Inclusion and exclusion criteria

Inclusion criteria were: 1) age 18–70 years; 2) continuous residence (> 4 years) in the geographic region of the study; 3) diagnosis of stable bronchial asthma according to GINA criteria[17]; 4) non-smoker; 5) no respiratory infections in the month prior to enrolment; 6) no oral corticosteroids in the last month; 7) no biological treatment with anti-IgE monoclonal antibodies; 8) no allergenic immunotherapy.

Exclusion criteria were: 1) pregnancy; 2) moderate to severe active alcohol use; 3) severe atopic dermatitis; 4) presence of any lung disease, autoimmune disease or systemic inflammatory disease, or cancer.

Control group

The control group consisted of healthy, non-smoking volunteers age 18 to 79 years, without rhinitis, allergic asthma, or other allergic symptoms (GINA criteria) or other lung disease. Controls were recruited from among staff members at our hospital. Participation was completely voluntarily. All controls were required to present a negative prick test for aeroallergens and Der p 1-specific IgE, and negative Phadiatop test.

Assessments and study procedures

Upon enrolment, demographic and clinical variables were assessed and recorded for all participants. On the same day, the following assessment were performed: Fe_NO (exhaled nitric oxide test); forced spirometry; inflammatory cell count in induced sputum; eosinophil count in peripheral blood; total serum IgE levels; and skin prick test for common aeroallergens. Patients also completed the validated Spanish-language version of the Asthma Control Test (ACT)[20].

Fe_NO was measured before spirometry using an electrochemical equipment (NO Vario Analyzer; FILT Lungen and Thorax Diagnostic GmbH, Berlin, Germany) and an expiratory maneuver providing a sustained 50 mL/s flow from total lung capacity, following the 2005 recommendations of the American Thoracic Society/European Respiratory Society[21]. Spirometry was performed using a Daptospir-600 spirometer (Sibelmed, S.A., Barcelona, Spain) in accordance with the 2003 recommendations of the Spanish Society of Pneumology and Thoracic Surgery (SEPAR), with FEV₁ (forced expiratory volume in 1 second) in the reference range when 80% of the predicted value [22,23].

Skin prick testing was performed according to standard procedures, with wheal diameters ≥ 4 mm considered positive. Allergic asthma was defined as the presence of asthma symptoms for one or more allergens, with positive skin prick tests for these allergens. Well-controlled asthma was defined as ACT ≥ 20.

Induction and analysis of sputum

Induced sputum was evaluated following the procedures previously described by our group[24] and by Pizzichini et al.[25]. Briefly, mucus plugs were manually selected and weighed, and incubated at room temperature for 15 min in 4 times the weight (in ml) of the selected plug (in mg) in 0.1% dithiothreitol (Calbiochem, San Diego, Calif., USA), washed with 4 times the plug weight (in ml) in Dulbecco’s PBS, and gravity filtered through a 41-μm-pore nylon net filter (Millipore Inc; Billerica, Mass., USA). Each specimen was homogenised and aliquoted into two equal volumes. A Neubauer hemocytometer was used to determine total cell count; visually identifiable squamous epithelial cells were not included in the total cell count. Samples with insufficient sputum cell numbers (<1000 × 10⁶ cells/g) were excluded. Cell viability was determined by light microscopic assessment using trypan blue exclusion staining. The cells underwent centrifugation to obtain a cell pellet and a supernatant. The cell pellet was used for differential cell counts (macrophages, eosinophils, neutrophils, lymphocytes, and bronchial epithelial cells) performed on May-Grünwald-Giemsa-stained preparations. A differential leukocyte analysis of nonsquamous cells (Diff-Quik stained) was performed on a minimum of 400 cells. Differential cell counts are expressed as the percentage of total nonsquamous nucleated cells. Reference values for the cell counts were performed as described in other publications[26].

Measurement of total and specific IgE antibodies

Total and specific IgE in induced sputum supernatants were measured by the ImmunoCAP fluoroenzyme immunoassay (Phadia ThermoFisher Scientific) following the manufacturer’s instructions. The test was considered positive at > 2kU/L for total IgE and > 0.35kU/L for specific IgE, according to manufacturer’s recommendation.

Statistical analysis

Categorical variables were expressed as absolute and relative frequencies and quantitative variables as mean and standard deviation (SD). Groups were compared using ANOVA or chi-square test as appropriate. Given that the distribution of total and specific IgE values in serum and sputum were not normal, non-parametric tests (Mann-Whitney or Kruskal-Wallis for independent samples) were applied; the values were expressed as medians with interquartile ranges and ranges. The significance values in the case of non-parametric tests were adjusted by the Bonferroni correction. For the correlation analyses, Spearman's Rho test was used. Statistical significance was set at p<0.05. Statistical analysis was performed with Statistical Package for the Social Sciences version 18.0 (SPSS, Chicago, IL).

Ethics approval and consent to participate

The study design complied with the principles of the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee at the Hospital de la Santa Creu i Sant Pau in Barcelona (COD 26/2012). All participants provided written informed consent. All patient-related data were anonymised, with the identity of the participating known only by the treating physicians. The clinicaltrials.gov identifier is NCT03640936.

Correlations

Using data from the whole sample (patients and controls), we calculated the correlations between total and Der p 1-specific IgE levels in sputum and serum. Table 3 shows a matrix with all significant correlations (p<0.0001) in descending order from strongest to weakest. As that table makes clear, the strongest correlation was between total IgE and Der p 1-specific IgE in serum.

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Table 3. Significant correlations between IgE values in serum and induced sputum.

https://doi.org/10.1371/journal.pone.0228045.t003

Correlation between IgE in sputum and serum

In the present study, we used the sputum induction and analysis methods described by Araujo et al.[31], who validated laboratory measurements of total and Der p 1-specific IgE in induced sputum supernatant versus serum levels, but in a heterogeneous sample involving patients with diverse pathologies, not only asthma. In recent years, several studies have correlated total IgE levels in induced sputum and serum, with sometimes contradictory findings[32]. In the present study, we found a highly significant correlation (rho, 0.498; p = 0.000) between total IgE levels in serum and sputum in our overall sample, a finding that is consistent with the results described by Manise et al. in asthmatic patients[28]. By contrast, other authors, including Mouthuy et al.[7] and Ahn et al[33] have not found any correlation between total IgE levels in sputum and serum. These contrasting findings raise further doubts about the sensitivity of the ImmunoCap technique used to measure IgE in sputum, potentially providing an additional explanation for the differences between our results and those of Mouthuy et al.

Data from a recent study conducted by Pillai et al.[34] could help to explain why we were unable to detect significant between-group differences in total IgE in induced sputum. Those authors suggest that IgE produced in the bronchial mucosa of nonatopic asthmatic patients may remain confined to the mucosa, bound to cells that carry those receptors. If this hypothesis is correct, it would explain why IgE is not readily detectable in induced sputum. Pillai and colleagues posited that both atopic and nonatopic asthmatics would have greater total IgE concentrations in the airways than in serum. To test this, they determined IgE levels in the blood and bronchial mucosa of 10 atopic and 10 nonatopic asthmatic patients and 10 nonatopic controls, finding that median total IgE levels were significantly elevated in both the atopic and nonatopic asthmatic patients versus controls. These data are consistent with the hypothesis that IgE synthesis, sequestration, or both are ongoing in the bronchial mucosa of both nonatopic and atopic asthmatic patients. Interestingly, Pillai et al. also suggest, as other several authors have previously proposed[10,35,36], that increased bronchial mucosal IgE production in nonatopic asthmatic patients may be directed against targets other than allergens, including possible “autoallergens”, or that there are allergen-independent roles for IgE in the pathophysiology of asthma.

Overall, the findings reported in studies which use more invasive but more sensitive methods (e.g., bronchial biopsy) strongly suggest the presence of elevated total IgE production in the airways of both allergic and nonallergic asthma patients[10,34–36]. The fact that we were unable to confirm the findings reported by Mouthuy et al. with regard to detecting significant differences in sputum IgE levels between nonatopic asthmatics and healthy controls, together with the contradictory data reported to date regarding the correlation between IgE levels in serum and induced sputum, suggests that more sensitive methods of measuring IgE in sputum may be required.

Total and Der p-specific IgE in induced sputum in healthy individuals

A secondary aim of this study was to determine, on a pilot basis, the standard levels of total and Der p 1-specific IgE in the induced sputum of healthy controls. We found the following values: median (IQR) total IgE in sputum in the controls was 3.16 (1.41) KUA/L. The Der p 1-specific values were 0.06 (0.02). Evidently, the small sample (n = 9) of healthy controls are insufficient to define standard levels, but these data provide an initial estimate. Nevertheless, more data from larger studies will be needed to confirm these initial levels, especially considering the small sample and the potential limitations in the assay technique used to measure IgE in the sputum supernatant.

Study strengths and limitations

The main limitation of the present study is the limited number of healthy controls. Another limitation may be related to the lack of significant differences between the groups in total IgE in induced sputum, which points to limitations in the measurement technique that may have influenced our findings. An important strength of the study is that this is, to our knowledge, only the second study conducted to date to specifically determine total and Der p-specific IgE in induced sputum of allergic, nonallergic, and healthy controls. Given the conflicting results of our study and those reported by Mouthuy et al., additional studies are needed. Finally, another important strength is the well-selected and well-defined sample of patients and controls; we used very strict diagnostic criteria (based on the most recent clinical guidelines) to define both allergic and nonallergic asthma, as well as for the healthy controls.