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Original Research: ASTHMA |

Changes in Sputum Eicosanoids and Inflammatory Markers After Inhalation Challenges With Occupational Agents FREE TO VIEW

Mar Fernández-Nieto, MD; Beatriz Sastre, BS; Joaquín Sastre, MD, PhD, FCCP; Carlos Lahoz, MD, PhD; Santiago Quirce, MD, PhD; Mauro Madero, MD; Victoria del Pozo, PhD
Author and Funding Information

Affiliations: From the Allergy Department (Drs. Fernández-Nieto, J. Sastre, and Madero) and Immunology Department (Drs. Lahoz and del Pozo, and Ms. B. Sastre), Fundación Jiménez Díaz Capio and Centro de Investigacíon Biomedica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; and the Allergy Department (Dr. Quirce), Hospital Universitario La Paz and CIBERES, Madrid, Spain.

Correspondence to: Joaquín Sastre, MD, PhD, FCCP, Allergy Department, Fundación Jiménez Díaz, Av Reyes Católicos 2, 28040 Madrid, Spain; e-mail: jsastre@fjd.es


Funding/Support: This study was supported by Red Respira C03/011, CIBERES, and Sociedad Española de Alergia e Inmu-nología Clinic (SEAIC), and by fellowship grants to Dr. Madero from the Conchita Rábago Foundation.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal.org/site/misc/reprints.xhtml).


© 2009 American College of Chest Physicians


Chest. 2009;136(5):1308-1315. doi:10.1378/chest.09-0103
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Background:  An increase in cysteinyl-leukotrienes (LTs) after specific inhalation challenge (SIC) with common allergens in patients with atopic asthma has been shown previously, but there are scarce data with occupational agents. We sought to determine whether there are differences in lower airway inflammatory markers and the production of cytokines and eicosanoids between patients with a positive or negative SIC response to occupational agents.

Methods:  Twenty-six patients with suspected occupational asthma and 13 healthy control subjects were studied. Spirometry, methacholine challenge, and sputum induction were performed at baseline and 24 h after SIC with occupational agents. Several cytokines and inflammatory mediators, including eicosanoids, were measured in sputum.

Results:  Twenty-six SICs were carried out with high-molecular-weight or low-molecular-weight agents, and the responses were positive in 18 patients. SIC elicited nine early asthmatic responses, two dual asthmatic responses, and seven isolated late asthmatic responses. Significant increments in sputum eosinophil counts were found only in patients with positive SIC responses compared with baseline values. Interleukin-10 levels were decreased in patients with positive and negative SIC responses compared to those in healthy control subjects. A significant increase (p < 0.05) in the LTC4/prostaglandin E2 (PGE2) ratio was observed in patients after positive SIC responses compared to those with negative SIC responses.

Conclusions:  Overexpression of LTC4, relative underproduction of PGE2, and greater airway eosinophilia were observed in patients with positive SIC responses.

Figures in this Article

Induced sputum is a useful, noninvasive tool for the diagnosis and monitoring of occupational asthma (OA).1 Patients with OA show an increase in the number of eosinophils in sputum at work compared with numbers on days off from work.2,3 Moreover, a significant increase in eosinophil counts in sputum following specific inhalation challenge (SIC) has been observed in patients with OA caused by high-molecular-weight (HMW) and low-molecular-weight (LMW) agents,49 but an increase in neutrophils also has been found,10,11 especially after exposure to diisocyanates. The increase in sputum eosinophils has been proposed15 as a sensitive marker of the bronchial response to occupational agents.

In the airway inflammatory process of OA, eosinophilia is associated with an increased number of T cells, especially lymphocyte subsets (CD4+/CD8+), which exhibit signs of activation.12,13 It has been suggested14 that CD8+ cells also are key in OA, with an IgE-independent mechanism.

Hallstrand et al15 reported an increase in the leukotriene (LT) C4/prostaglandin E2 (PGE2) ratio in sputum in response to exercise challenge in patients with asthma. In patients with isocyanate-induced asthma, Lemiere et al16 found an increase in sputum LTB4 and interleukin (IL)-8 after exposure to isocyanates compared with baseline levels, whereas no increase was found in LTC4. In this study, we assessed changes in the LTC4 and PGE2 concentrations in induced sputum before and after SIC with occupational agents in a group of patients with suspected OA induced by HMW or LMW agents as well as changes in sputum cell counts and cytokine expression.

Subjects

Twenty-six patients with suspected OA caused by HMW or LMW occupational agents and 13 healthy control subjects were enrolled in the study. All subjects were recruited from the Fundación Jimenez Díaz Allergy Clinic (Madrid, Spain). The subjects' clinical characteristics are shown in Table 1.

Table Graphic Jump Location
Table 1 Clinical Characteristics of Subjects

Values are presented as the median (range) or No. (%), unless otherwise indicated. ND = not done.

*PC20 > 16 mg/mL.

†Geometric mean.

Study Protocol

On the first day (control day), a full medical and occupational history was completed. For patients who were receiving therapy with inhaled corticosteroids (n = 26), the drugs were withdrawn for at least 1 week before sputum induction. No patient was receiving LT modifiers or nonsteroidal anti-inflammatory drugs. Spirometry and methacholine challenge were performed as described elsewhere.17 These tests, together with sputum induction, were performed at baseline and 24 h after SIC with occupational agents. This study was approved by our Institutional Clinical Trials and Ethics Committee, and all subjects gave written informed consent to participate in the study.

SICs

SIC with LMW agents was performed as previously described.7,18 SIC with HMW agents was carried out following the tidal breathing inhalation method as previously described.19

Sputum Induction and Processing

Sputum was induced with inhalations of increasing concentrations (3%, 4%, and 5%) of hypertonic saline solution,20 and was processed and examined for nonsquamous cell counts, as previously described,21 and following European Respiratory Society22 recommendations. Solid sputum material was selected from saliva, divided, and processed within 2 h by two parallel procedures. The protocol used was previously described23 and slightly modified. Sputum was treated with a 0.1% dithiothreitol (DTT) solution to improve cell dispersion. Sputum was treated with a similar volume of DTT and incubated on a shaker at 37° C for 15 min. To prevent the effect of DTT on the cell suspension, an equal volume of Dulbecco phosphate-buffered saline (PBS) solution was added. Mucus was then removed by means of filtration through a 100-μm-pore nylon mesh, and the filtrate was centrifuged at 400g for 10 min to sediment any cells present. The supernatant was aliquoted and stored at −80°C until analysis. The pellet was resuspended in a PBS solution, bovine serum albumin, and ethylenediaminetetraacetic acid wash. The total cell count, viability, and percentage of squamous cells were determined by trypan blue exclusion staining. The pellet was adjusted for 1 × 106 cells/mL with a PBS solution containing 2% fetal calf serum for flow cytometry. The rest of the cell suspension was resuspended (TRIzol; Invitrogen; Carlsbad, CA) and stored at −80°C until used.

Flow Cytometry of Sputum Cells

For each test, 100 μL of sputum cell suspension was incubated with 3 μL of each monoclonal antibody (BD Biosciences; San Jose, CA) for 30 min at 4°C in the dark. Cells were washed with PBS solution containing 2% fetal calf serum and resuspended in 400 μL of PBS solution containing 2% fetal calf serum. Subsequently, the cells were identified with a two-laser, five-parameter flow cytometer (FACSCalibur; BD Biosciences) and analyzed using computer software (Cell Quest; BD Biosciences). Typically, 10,000 to 50,000 total cells were collected and analyzed per sample. All sputum cell samples were stained with phycoerythrin (PE)-Cy5-conjugated antihuman CD45 monoclonal antibody to exclude all nonleukocyte cells in each test. The panel consisted of CCR3 fluorescein isothiocyanate (FITC)/CD16 PE, CCR3 FITC/DR PE, CD69 FITC/CD16 PE, CD14 FITC/DR PE, CD4 FITC/CD2 PE, CD4 FITC/CD25 PE, CD19 FITC/CD2 PE, CD34 FITC/CDw125 PE, CD63 FITC/IgE PE, and isotype control (BD Biosciences), which were used to establish background fluorescence for each fluorochrome.

Measurement of Soluble Mediators, Cytokines, and Eicosanoids

We analyzed IL-5, IL-10, IL-1, IL-8, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay (or ELISA) kits (Bender MedSystems GmbM; Vienna, Austria). The detection limit of these assays was between 0.99 and pg/ml (1.45, 0.99, 1.06, 5, 0.99, and 3.83 pg/mL, respectively). All these assays have different coefficient of variations for intraassay (6.6%, 3.2%, 5.4%, 4.1%, 4.5%, and 6.9%, respectively) and interassay (6.8%, 5.6%, < 10%, 10.9%, 5.7%, and 7.4%, respectively). Moreover, we tested two types of lipidic mediators, PGE2 and LTC4, by enzyme-linked immunosorbent assay (Cayman Chemical Company; Ann Arbor, MI). Previously, to test lipid mediators, sputa were treated to remove protein and other contaminants that could interfere with the assay. In all subjects, mediators were measured in neat supernatants in duplicate. PGE2 assay shows a detection limit of 2 pg/mL, with a coefficient of variation for intraassay values of 5% and a coefficient of variation for interassay values of 4%, with a specificity of 100%. The LTC4 assay has a detection limit of 13 pg/mL, a specificity of 100%, and a coefficient of variation for intraassay values and interassay values of 8% and 10%, respectively.

We developed several experiments to analyze a possible effect of DTT in these kinds of experiments. We observed that this factor does not exercise any effect in the evaluation of lipid mediators. This conclusion has been observed by others.24,25

Statistical Analysis

Subject characteristics were described with descriptive statistics and expressed as means and SDs. All measures were performed in a blinded fashion. Comparisons of mediator concentration and sputum-different cell counts across the three groups and between the groups were performed with Kruskal-Wallis test and Mann-Whitney U test, and analysis of variance and unpaired t test with Welch correction for nonparametric data and parametric data, respectively. Normality was analyzed with the Kolmogorov-Smirnov test. A difference was considered to be significant when p was ≤ 0.05. Statistical analyses were performed with a statistical software package (GraphPad InStat3; GraphPad Software Inc; San Diego, CA).

SICs

Twenty-six SICs were carried out and elicited nine early asthmatic responses, two dual asthmatic responses, and seven isolated late asthmatic responses. The implicated occupational agents were LMW agents, including isocyanates (n = 7), eugenol (n = 1), formaldehyde (n = 1), and HMW agents, including latex (n = 3), tampico fiber (n = 1), wheat flour (n = 3), fungal α-amylase (n = 1), and esparto grass fiber (n = 1). The eight subjects with negative SIC responses were exposed to LMW agents, including isocyanates (n = 1), formaldehyde (n = 1), styrene (n = 1), cutting mineral oil (n = 1), wood (n = 1), and glutaraldehye (n = 1), and an HMW agent, latex (n = 2).

All patients with positive SIC responses but one had a positive methacholine test result at baseline, and seven patients had a twofold or greater increase in provocative concentration of a substance (methacholine) causing a 20% fall in FEV1 (PC20) that was observed 24 h after SIC. In five patients with negative SIC responses, methacholine PC20 was > 16 mg/mL, and none changed after SIC.

Differential Sputum Cell Counts

Fifty-two sputum samples from patients with positive and negative SIC responses and healthy control subjects were analyzed. Sputum samples had < 10% squamous epithelial cells and > 85% cell viability (Table 2).

Table Graphic Jump Location
Table 2 Total and Differential Cell Counts in Induced Sputum (Flow Cytometry)

Results are expressed as the median (range).

*p < 0.05 compared with healthy control subjects.

†p < 0.01 compared with healthy control subjects.

‡p < 0.05 compared with OA after SIC.

Macrophage counts showed differences between a control group (55.17%) and the other groups (negative SIC responses, 17.52%; positive SIC responses, 28.7% at baseline and 32.27% post-SIC; p < 0.05). In the case of neutrophils, patients with positive SIC responses both at baseline and post-SIC showed a significantly higher percentage than healthy control subjects (63.37% and 62.15% vs 38.34%, respectively; p < 0.01 and p < 0.05, respectively); the percentage of this cellular type in patients with negative SIC responses was similar to that in healthy control subjects. Figure 1B shows the individual variations in the percentage of neutrophils after SIC.

Figure Jump LinkFigure 1 A: changes in sputum eosinophils at baseline and after SIC in patients with positive and negative SIC responses. B: changes in sputum neutrophils at baseline and after SIC in patients with positive and negative SIC responses.Grahic Jump Location

Induced sputum eosinophil percentages were significantly higher (p < 0.05) in all patients with suspected OA (patients with negative and positive SIC responses) than in the healthy control subjects (Table 2). Moreover, eosinophils significantly increased in patients after positive SIC responses compared to baseline levels (median, 3.19% and 4.15%, respectively; p < 0.05) [Fig 1A, Table 2]. The statistical analyses were repeated, excluding the two patients who were smokers, and the results did not change.

In two patients with negative SIC responses, an increase in sputum eosinophils (9.92% and 8.25%) was observed after challenge. One of them had a negative methacholine test result before and after challenge with styrene. Thus, this patient was given a diagnosis of occupational eosinophilic bronchitis. Moreover, the baseline PGE2 level in sputum for this patient was 17 pg/mL, and after the challenge it increased to 201 pg/mL, which is in keeping with our previous results26 in patients with eosinophilic bronchitis. In the other patient, the methacholine test was positive and did not change after SIC with pine wood (PC20, 5.5 vs 3.3 mg/mL, respectively). Because the methacholine test result was positive, the patient could not be given a diagnosis of eosinophilic bronchitis, and because no asthmatic response was observed, he could not be given a diagnosis of OA due to pine wood. An increase in sputum eosinophils after SIC in patients who did not respond to SIC has been reported previously,27 and its clinical relevance is uncertain.

Cytokine Levels in Induced Sputum Supernatant From Patients With and Without OA and Healthy Control Subjects

We assessed the following cytokine levels: IL-5, IL-10, and IFN-γ, and representatives of T helper (Th) type 2 and Th1 cytokines. Moreover, we measured proinflammatory cytokines such as IL-1, IL-8, and TNF-α. The results indicated that IL-5 levels were increased in the patients with positive SIC responses (at baseline and 24 h after SIC) compared with those seen in healthy control subjects (p < 0.01) [Fig 2].

Figure Jump LinkFigure 2 Sputum fluid-phase measurement of cytokines from healthy control subjects and patients with negative and positive SIC responses. Levels of IL-5, IL-10, and IFN-γ in sputum from different groups of subjects were determined by enzyme-linked immunosorbent assay. Significant differences in IL-5 (** = p < 0.01) levels were obtained for patients with positive SIC responses vs healthy control subjects. Additionally, lower IL-10 levels were detected in patients with negative SIC and positive SIC responses with respect to healthy control subjects (* = p < 0.05).Grahic Jump Location

With respect to the production of regulatory cytokines by sputum cells, IL-10 concentrations were significantly lower in induced sputum samples from patients with suspected OA (with either positive or negative SIC responses) compared with healthy control subjects (Fig 2) [p < 0.05]. Levels were very similar before and after positive SIC responses, but they were more elevated in patients with negative SIC responses. Moreover, there were no significant changes in the levels of IFN-γ between both groups with suspected OA and the control group (Fig 2). Otherwise, analysis of the expression of several cytokines in the study groups showed that IL-5 is the predominant mediator in the local inflammatory process in the airways, with a Th2 cytokine profile in both diseases.

We observed that IL-1 and TNF-α had a similar profile, although statistically significant changes were not identical. IL-1 levels were significantly lower in patients with negative SIC responses than in those 24 h after positive SIC response and healthy control subjects (7.86 pg/mL vs 26.11 and 34.11 pg/mL, respectively; p < 0.01 and p < 0.001). TNF-α levels were significantly higher in healthy control subjects than in patients with suspected OA (positive and negative SIC responses) at baseline (30.51 pg/mL vs 3.83 and 17.63 pg/mL, respectively; p < 0.05) [Fig 3, top].

Figure Jump LinkFigure 3 Inflammatory cytokine levels in induced sputum supernatants. Top: a comparison of IL-1 and TNF-α levels in induced sputum supernatants among the study groups. Bottom: a comparison of IL-8 levels among the study groups in induced sputum supernatants. Sputum from patients with negative SIC responses had lower levels than healthy control subjects and patients with positive SIC responses. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001.Grahic Jump Location

IL-8 concentration was significantly elevated in patients with positive SIC responses compared with patients with negative SIC responses (526.64 and 383.39 pg/mL, respectively; p < 0.05) under baseline conditions. In patients with positive SIC responses, IL-8 concentration 24 h after challenge (129.9 pg/mL) was significantly higher than in those with negative SIC responses at baseline (p < 0.05) [Fig 3, bottom] but not in patients with positive SIC responses at baseline.

Eicosanoids in Induced Sputum Supernatant

An inverse relationship was observed between PGE2 and LTC4 concentrations in patients with positive SIC and negative SIC responses. Thus, the LTC4/PGE2 ratio in induced sputum supernatant was significantly increased in patients with positive SIC responses 24 h after challenge compared with patients with negative SIC responses, the difference being more than twofold (p < 0.05) [Fig 4]. There were no intergroup differences in sputum cells count, cytokines, or eicosanoid measurements in patients with positive SIC responses when they were subdivided by either the causative agent (HMW or LMW agent) or the type of asthmatic reaction (immediate or late).

Figure Jump LinkFigure 4 LTC4/PGE2 ratio in induced sputum from healthy control subjects and patients with negative and positive SIC responses. Data are represented as the geometric mean ± SD. Patients with a positive SIC response had a significantly higher LTC4/PGE2 ratio than patients with a negative SIC response.Grahic Jump Location

We observed several changes in sputum inflammatory markers and eicosanoids after inhalation challenges with occupational agents. An increase in inflammatory parameters associated with eosinophilic infiltration (sputum eosinophils and IL-5) was found, which was in keeping with the results observed by other authors4,5 of sputum cells before and after SIC with occupational agents. In the majority of cases of OA, the percentage of sputum eosinophils increased after exposure to occupational agents in the laboratory or at the workplace compared with baseline, but an increase in sputum neutrophils also has been observed. In agreement with this finding, we observed a significant increase in IL-8 concentration but not in neutrophils in patients with positive SIC responses compared with patients with negative SIC responses. Lemiere et al4 documented increases in both eosinophils and neutrophils in sputum after asthmatic reactions induced by HMW and LMW agents, and these cellular changes occurred independently from the temporal pattern of asthmatic reactions. Di Franco et al28 found more neutrophils and fewer eosinophils in the sputum of patients with OA caused by LMW agents than in that of patients with HMW agent-induced OA or non-OA. These findings indicate that neutrophils are involved in OA, perhaps more frequently but not exclusively, when OA is provoked by LMW agents. Therefore, OA may be subdivided into eosinophilic or neutrophilic asthma phenotypes.29

The use of sputum cell counts has been shown to be useful in the investigation of OA, and performing flow cytometry on sputum cell counts may allow the identification of cell populations that otherwise might not be identified, which is especially important for basophils. In a previous case report,7 we demonstrated a small increase in sputum eosinophils but a large increase in activated basophils 24 h after exposure to styrene along with functional changes supporting the diagnosis of OA due to styrene. An increase in basophil counts in induced sputum has been found30,31 in patients with atopic asthma and in patients with OA after SIC. In this study, we did not observe significant changes in the number of activated basophils in the whole group after positive SIC response, but there was an important increase (4.85-fold) in activated basophils in six patients after positive SIC response.

Classically, allergic asthma and rhinitis are described as associated with a Th2 activation. However, recent work has indicated that Th1 activation also can be associated with these diseases due to a defect in regulatory T-cell activation. OA and occupational rhinitis are peculiar cases of these diseases in which the T-cell activation profile is unclear. Previous studies32,33 in mice have demonstrated a Th2 cell activation, with an increase in IL-5 and IL-4 production in experimental OA. Other mouse studies have found a Th1 activation associated with the Th2 activation previously described.13,34 In addition, in non-OA, some evidence35 indicates that Th1 and Th2 cell activation could be concomitantly associated with a decrease in the regulatory T-cell frequency and activation, and it has been described36 that in patients with OA and occupational rhinitis the proportion of peripheral regulatory T cells decrease after SIC. In our study, analysis of the expression of several cytokines showed that IL-5 (a Th2 cytokine) is the predominant mediator in the local inflammatory process in the airways, with a Th2 cytokine profile in the patients with suspected OA studied. We observed that IL-1 levels were significantly lower in patients with negative SIC responses than in patients with positive SIC responses and healthy control subjects. By contrast, TNF-α levels were significantly higher in healthy control subjects than in patients with negative SIC responses and patients with positive SIC responses at baseline.

To date, controversial data about the cytokines involved in OA in response to SIC have been published. In vitro experiments have reported that peripheral blood mononuclear cells of workers with diisocyanate-induced OA demonstrated a hapten-specific production of histamine-releasing factor37 and the release of monocyte chemoattractant protein-1, IL-8, TNF-α, and IFN-γ.14 Interestingly, in our study, IL-10 levels were decreased in patients with asthma with suspected OA with either negative or positive SIC responses. IL-10 is a cytokine with broad anti-inflammatory properties, and it has been shown38,39 to play an important role in the regulation of Th2 cell responses. Therefore, IL-10 is a potent anti-inflammatory cytokine that inhibits various types of inflammatory disorders. In this regard, IL-10 produced by either Th1 or Th2 cells may be an important feedback regulator controlling the damage associated with an exaggerated inflammatory response. It is likely that constitutive production of IL-10, as occurs in the lungs of healthy persons, contributes to maintaining a balance that inhibits inflammation. Conversely, decreased or absent IL-10, as occurs in OA, may contribute to a regulatory imbalance in which inflammatory responses could become pathologic. Asthma and fatal ARDS also have been associated with decreased IL-10 production,40,41 suggesting the importance of this cytokine in human lung diseases.

Another important issue in the immunopathology of OA is the relationship between sputum PGE2 and LTs before and after SIC. In our study, the LTC4/PGE2 ratio in induced sputum supernatant was significantly increased in patients with positive SIC responses 24 h after the challenge compared with patients with negative SIC responses. The cyclooxygenase product PGE2 is produced by several cells in human airways, including epithelium42 and smooth muscle43; it has inhibitory effects on inflammatory cells and bronchoprotective effects in patients with bronchial asthma. PGE2 has been shown to protect against exercise-induced bronchoconstriction,44 allergen-induced bronchoconstriction,45 and aspirin-induced bronchoconstriction,46 as well as against bronchoconstrictor agents such as methacholine and histamine.47 We recently have shown26 that sputum PGE2 concentration is significantly higher, and LTC4/PGE2 ratio is lower in patients with nonasthmatic eosinophilic bronchitis than in patients with asthma and control subjects. One of the patients with a negative SIC response fulfilled the criteria of occupational eosinophilic bronchitis induced by styrene. Moreover, the patient showed a 10-fold increase in sputum PGE2 concentration after challenge, which is in agreement with our previous investigations.26 The disregulated synthesis of bronchoconstrictor (LTC4) and bronchoprotective (PGE2) eicosanoids may explain the abnormal airway function found in asthma.

There are scarce data in the literature on this issue. Similar results have been reported by Macfarlane et al25 that demonstrated an increase in cysteinyl-LTs (cysLTs) but not in the prostanoids PGD2 and PGE2 associated with a late asthmatic reaction after allergen challenge in patients with atopic asthma and that have been described15 in patients with exercise-induced bronchoconstriction. In contrast, Lemiere et al16 showed a significant increase in cysLT1 and BLT1 receptor expression as well as a release of LTB4 and IL-8 after exposure to isocyanates compared with the baseline only in patients with isocyanate-induced asthma, but their data showed no increase in LTC4.

The alteration in the cysLTs/PGE2 ratio identified in the present study may be indicative of airway epithelial injury. Taken together, these data suggest that the relative underproduction of PGE2 and overproduction of cysLTs also may be important factors in the bronchoconstrictive response induced by occupational agents. In conclusion, we have demonstrated for the first time, to our knowledge, that the LTC4/PGE2 ratio in induced sputum is increased in patients after positive SIC responses to occupational agents compared with patients with negative SIC responses.

cysLT

cysteinyl-leukotriene

DTT

dithiothreitol

FITC

fluorescein isothiocyanate

HMW

high molecular weight

IFN

interferon

IL

interleukin

LMW

low molecular weight

LT

leukotriene

OA

occupational asthma

PBS

phosphate-buffered saline

PC20

provocative concentration of a substance causing a 20% fall in FEV1

PE

phycoerythrin

PGE2

prostaglandin E2

SIC

specific inhalation challenge

Th

T helper

TNF

tumor necrosis factor

Author contributions: Drs. Fernández-Nieto, J. Sastre, Quirce, and Madero participated in the selection of patients and clinical studies. Drs. Lahoz and del Pozo, and Ms. B. Sastre performed all laboratory work.

Financial/nonfinancial disclosures: Dr. J. Sastre reports having served as a consultant to Phadia, Schering-Plough, and GlaxoSmithKline; having been paid lecture fees by Novartis, GlaxoSmithKline, Stallergenes, and UCB; and having received grant support from Phadia, GlaxoSmithKline, and ALK-Abelló. Drs. Fernández-Nieto, Lahoz, Quirce, Madero, and del Pozo, and Ms. B. Sastre have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

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Efthimiadis A, Spanevello A, Hamid Q, et al. Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation. Eur Respir J. 2002;20suppl 37:19s-23s
 
Hadjicharalambous C, Dent G, May RD, et al. Measurement of eotaxin (CCL11) in induced sputum supernatants: validation and detection in asthma. J Allergy Clin Immunol. 2004;113:657-662. [PubMed]
 
Pavord ID, Ward R, Woltmann G, et al. Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med. 1999;160:1905-1909. [PubMed]
 
Macfarlane AJ, Dworski R, Sheller JR, et al. Sputum cysteinyl leukotrienes increase 24 h after allergen inhalation in atopic asthmatics. Am J Respir Crit Care Med. 2000;161:1553-1558. [PubMed]
 
Sastre B, Fernández-Nieto M, Mollá R, et al. Increased prostaglandin E2 levels in the airway of patients with eosinophilic bronchitis. Allergy. 2008;63:58-66. [PubMed]
 
Obata H, Dittrick M, Chan H, et al. Sputum eosinophils and exhaled nitric oxide during late asthmatic reaction in patients with western red cedar asthma. Eur Respir J. 1999;13:489-495. [PubMed]
 
Di Franco A, Vagaggini B, Bacci E, et al. Leukocyte counts in hypertonic saline-induced sputum in subjects with occupational asthma. Respir Med. 1998;92:550-557. [PubMed]
 
Wanzel SE. Asthma: defining of the persistent adult phenotypes. Lancet. 2006;368:804-813. [PubMed]
 
Gauvreau GM, Lee JM, Watson RM, et al. Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics. Am J Respir Crit Care Med. 2000;161:1473-1478. [PubMed]
 
Krakowiak A, Krawczyk-Adamus P, Dudek W, et al. Changes in cellular and biochemical profiles of induced sputum after allergen-induced asthmatic response: method for studying occupational allergic airway inflammation. Int J Occup Med Environ Health. 2005;18:27-33. [PubMed]
 
Maestrelli P, Occari P, Turato G, et al. Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI). Clin Exp Allergy. 1997;27:1291-1298
 
Hardy CL, Kenins L, Drew AC, et al. Characterization of a mouse model of allergy to a major occupational latex glove allergen Hev b 5. Am J Respir Crit Care Med. 2003;167:1393-1399. [PubMed]
 
Herrick CA, Xu L, Wisnewski AV, et al. A novel mouse model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge. J Allergy Clin Immunol. 2002;109:873-878. [PubMed]
 
Karagiannidis C, Akdis M, Holopainen P, et al. Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol. 2004;114:1425-1433. [PubMed]
 
Mamessier E, Milhe F, Guillot C, et al. T-cell activation in occupational asthma and rhinitis. Allergy. 2007;62:162-169. [PubMed]
 
Herd ZL, Bernstein DI. Antigen-specific stimulation of histamine releasing factors in diisocyanate-induced occupational asthma. Am J Respir Crit Care Med. 1994;150:988-994. [PubMed]
 
Moore KW, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683-765. [PubMed]
 
Hawrylowicz CM. Regulatory T cells and IL-10 in allergic inflammation. J Exp Med. 2005;202:1459-1463. [PubMed]
 
Borish L, Aarons A, Rumbyrt J, et al. Interleukin-10 regulation in normal subjects and patients with asthma. J Allergy Clin Immunol. 1996;97:1288-1296. [PubMed]
 
Donnelly SC, Strieter RM, Reid PT, et al. The association between mortality rates and decreased concentrations of interleukin-10 and interleukin-1 receptor antagonist in the lung fluids of patients with the adult respiratory distress syndrome. Ann Intern Med. 1996;125:191-196. [PubMed]
 
Churchill L, Chilton FH, Resau JH, et al. Cyclo-oxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells. Am Rev Respir Dis. 1989;140:449-459. [PubMed]
 
Delamere F, Holland E, Patel S, et al. Production of PGE2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo-oxygenase inhibitors. Br J Pharmacol. 1994;111:983-988. [PubMed]
 
Melillo E, Woolley KL, Manning PJ, et al. Effect of inhaled PGE2 on exercise-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med. 1994;149:1138-1141. [PubMed]
 
Pavord ID, Wong CS, Williams J, et al. Effect of inhaled prostaglandin E2 on allergen-induced asthma. Am Rev Respir Dis. 1993;148:87-90. [PubMed]
 
Mastalerz L, Sanak M, Gawlewicz-Mroczka A, et al. Prostaglandin E2 systemic production in patients with asthma with and without aspirin hypersensitivity. Thorax. 2008;63:27-34. [PubMed]
 
Manning PJ, Lane CG, O'Byrne PM. The effect of oral prostaglandin E1 on airway responsiveness in asthmatic subjects. Pulm Pharmacol. 1989;2:121-124. [PubMed]
 

Figures

Figure Jump LinkFigure 1 A: changes in sputum eosinophils at baseline and after SIC in patients with positive and negative SIC responses. B: changes in sputum neutrophils at baseline and after SIC in patients with positive and negative SIC responses.Grahic Jump Location
Figure Jump LinkFigure 2 Sputum fluid-phase measurement of cytokines from healthy control subjects and patients with negative and positive SIC responses. Levels of IL-5, IL-10, and IFN-γ in sputum from different groups of subjects were determined by enzyme-linked immunosorbent assay. Significant differences in IL-5 (** = p < 0.01) levels were obtained for patients with positive SIC responses vs healthy control subjects. Additionally, lower IL-10 levels were detected in patients with negative SIC and positive SIC responses with respect to healthy control subjects (* = p < 0.05).Grahic Jump Location
Figure Jump LinkFigure 3 Inflammatory cytokine levels in induced sputum supernatants. Top: a comparison of IL-1 and TNF-α levels in induced sputum supernatants among the study groups. Bottom: a comparison of IL-8 levels among the study groups in induced sputum supernatants. Sputum from patients with negative SIC responses had lower levels than healthy control subjects and patients with positive SIC responses. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001.Grahic Jump Location
Figure Jump LinkFigure 4 LTC4/PGE2 ratio in induced sputum from healthy control subjects and patients with negative and positive SIC responses. Data are represented as the geometric mean ± SD. Patients with a positive SIC response had a significantly higher LTC4/PGE2 ratio than patients with a negative SIC response.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 Clinical Characteristics of Subjects

Values are presented as the median (range) or No. (%), unless otherwise indicated. ND = not done.

*PC20 > 16 mg/mL.

†Geometric mean.

Table Graphic Jump Location
Table 2 Total and Differential Cell Counts in Induced Sputum (Flow Cytometry)

Results are expressed as the median (range).

*p < 0.05 compared with healthy control subjects.

†p < 0.01 compared with healthy control subjects.

‡p < 0.05 compared with OA after SIC.

References

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Efthimiadis A, Spanevello A, Hamid Q, et al. Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation. Eur Respir J. 2002;20suppl 37:19s-23s
 
Hadjicharalambous C, Dent G, May RD, et al. Measurement of eotaxin (CCL11) in induced sputum supernatants: validation and detection in asthma. J Allergy Clin Immunol. 2004;113:657-662. [PubMed]
 
Pavord ID, Ward R, Woltmann G, et al. Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med. 1999;160:1905-1909. [PubMed]
 
Macfarlane AJ, Dworski R, Sheller JR, et al. Sputum cysteinyl leukotrienes increase 24 h after allergen inhalation in atopic asthmatics. Am J Respir Crit Care Med. 2000;161:1553-1558. [PubMed]
 
Sastre B, Fernández-Nieto M, Mollá R, et al. Increased prostaglandin E2 levels in the airway of patients with eosinophilic bronchitis. Allergy. 2008;63:58-66. [PubMed]
 
Obata H, Dittrick M, Chan H, et al. Sputum eosinophils and exhaled nitric oxide during late asthmatic reaction in patients with western red cedar asthma. Eur Respir J. 1999;13:489-495. [PubMed]
 
Di Franco A, Vagaggini B, Bacci E, et al. Leukocyte counts in hypertonic saline-induced sputum in subjects with occupational asthma. Respir Med. 1998;92:550-557. [PubMed]
 
Wanzel SE. Asthma: defining of the persistent adult phenotypes. Lancet. 2006;368:804-813. [PubMed]
 
Gauvreau GM, Lee JM, Watson RM, et al. Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics. Am J Respir Crit Care Med. 2000;161:1473-1478. [PubMed]
 
Krakowiak A, Krawczyk-Adamus P, Dudek W, et al. Changes in cellular and biochemical profiles of induced sputum after allergen-induced asthmatic response: method for studying occupational allergic airway inflammation. Int J Occup Med Environ Health. 2005;18:27-33. [PubMed]
 
Maestrelli P, Occari P, Turato G, et al. Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI). Clin Exp Allergy. 1997;27:1291-1298
 
Hardy CL, Kenins L, Drew AC, et al. Characterization of a mouse model of allergy to a major occupational latex glove allergen Hev b 5. Am J Respir Crit Care Med. 2003;167:1393-1399. [PubMed]
 
Herrick CA, Xu L, Wisnewski AV, et al. A novel mouse model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge. J Allergy Clin Immunol. 2002;109:873-878. [PubMed]
 
Karagiannidis C, Akdis M, Holopainen P, et al. Glucocorticoids upregulate FOXP3 expression and regulatory T cells in asthma. J Allergy Clin Immunol. 2004;114:1425-1433. [PubMed]
 
Mamessier E, Milhe F, Guillot C, et al. T-cell activation in occupational asthma and rhinitis. Allergy. 2007;62:162-169. [PubMed]
 
Herd ZL, Bernstein DI. Antigen-specific stimulation of histamine releasing factors in diisocyanate-induced occupational asthma. Am J Respir Crit Care Med. 1994;150:988-994. [PubMed]
 
Moore KW, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683-765. [PubMed]
 
Hawrylowicz CM. Regulatory T cells and IL-10 in allergic inflammation. J Exp Med. 2005;202:1459-1463. [PubMed]
 
Borish L, Aarons A, Rumbyrt J, et al. Interleukin-10 regulation in normal subjects and patients with asthma. J Allergy Clin Immunol. 1996;97:1288-1296. [PubMed]
 
Donnelly SC, Strieter RM, Reid PT, et al. The association between mortality rates and decreased concentrations of interleukin-10 and interleukin-1 receptor antagonist in the lung fluids of patients with the adult respiratory distress syndrome. Ann Intern Med. 1996;125:191-196. [PubMed]
 
Churchill L, Chilton FH, Resau JH, et al. Cyclo-oxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells. Am Rev Respir Dis. 1989;140:449-459. [PubMed]
 
Delamere F, Holland E, Patel S, et al. Production of PGE2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo-oxygenase inhibitors. Br J Pharmacol. 1994;111:983-988. [PubMed]
 
Melillo E, Woolley KL, Manning PJ, et al. Effect of inhaled PGE2 on exercise-induced bronchoconstriction in asthmatic subjects. Am J Respir Crit Care Med. 1994;149:1138-1141. [PubMed]
 
Pavord ID, Wong CS, Williams J, et al. Effect of inhaled prostaglandin E2 on allergen-induced asthma. Am Rev Respir Dis. 1993;148:87-90. [PubMed]
 
Mastalerz L, Sanak M, Gawlewicz-Mroczka A, et al. Prostaglandin E2 systemic production in patients with asthma with and without aspirin hypersensitivity. Thorax. 2008;63:27-34. [PubMed]
 
Manning PJ, Lane CG, O'Byrne PM. The effect of oral prostaglandin E1 on airway responsiveness in asthmatic subjects. Pulm Pharmacol. 1989;2:121-124. [PubMed]
 
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