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Keywords:

  • airway inflammation;
  • ECP;
  • induced sputum;
  • occupational asthma;
  • tryptase

Abstract

  1. Top of page
  2. Abstract
  3. Case report
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Background: Occupational asthma (OA) can be a debilitating disease even when removal from the workplace is achieved. Today, the “gold standard” in the assessment of OA is the bronchial provocation test (BPT). Induced sputum is a noninvasive method of exploring airway inflammation which can provide additional information about such challenges and thus could be applied in OA diagnosis and monitoring.

Methods: We report the study carried out in a grain worker sensitized to Lepidoglyphus destructor (Ld), who suffered from mild asthma at the workplace. Skin prick test and specific serum IgE were measured. Ld-BPT was performed, and the changes in eosinophil rates, and ECP and tryptase levels in induced sputum were studied 30 min and 18 h after Ld-BPT. We also determined the changes in nonspecific bronchial hyperresponsiveness (NSBH), given as PD20 values. To assess the specificity of the changes, we also carried out sputum induction and methacholine challenge after barley-BPT.

Results: An isolated immediate response was obtained with Ld-BPT, while barley-BPT was negative. Induced sputum showed higher tryptase levels 30 min after Ld-BPT, and higher eosinophil and epithelial cell percentages and ECP levels 18 h after Ld-BPT. There was also a decrease in methacholine PD20 values after Ld-BPT. Those changes were not observed after barley-BPT.

Conclusions: The study of eosinophilic and mast-cell markers in induced sputum provides additional knowledge about the inflammatory process occurring in the airways, suggesting that the study of induced sputum should be considered in the assessment of OA.

Bronchial inflammation is a major feature of asthmatic airways. However, the diagnosis of occupational asthma (OA) is made according to several algorithms (1, 2) based on clinical history, skin tests, specific IgE measurement, and lung-function tests such as airway obstruction reversibility, peak flow rate (PEFR) fluctuations, nonspecific bronchial hyperresponsiveness (NSBH) assessment, and the specific bronchial provocation test (BPT). OA can lead to permanent disability in spite of removal from exposure to the occupational allergen. Thus, early diagnosis and removal from exposure should be stressed.

During the last 6 years, several reports have demonstrated that induced sputum is a reproducible and valid method to evaluate bronchial inflammation in asthma (3, 4). This paper aimed to report a study on a cereal worker suffering from mild asthma at work. We tested whether, in the early stages of OA, the inhalation of the causative agent can induce airway inflammatory changes that can be detected by induced sputum analysis, and whether the assessment of such modifications in sputum might give additional information to that obtained from lung-function parameters in the evaluation of the response to allergen-BPT. We present the case report of a mill worker, sensitized to Lepidoglyphus destructor (Ld), in whom sputum eosinophil and epithelial cell percentages, and sputum ECP and tryptase levels increased after allergen-BPT.

Case report

  1. Top of page
  2. Abstract
  3. Case report
  4. Material and methods
  5. Results
  6. Discussion
  7. References

A 30-year-old man with no personal or family atopy antecedents, with the exception of his smoking habit (5 packs per year), is presented. He had been working at a silo for 10 years. He handled several cereals and other materials at work, including wheat, barley, malt, soybean, corn, sunflower seeds, alfalfa, and beet. His workplace was spacious and well ventilated. He did not use protective devices in his work. During the last 8 years and mainly when he worked with barley, he immediately developed symptoms consisting of contact urticaria and rhinitis. During the last 2 years, he also presented lower respiratory tract symptoms consisting of cough, wheezing, and chest tightness, which disappeared when exposure at the workplace stopped. He had no problem when eating cereals. Physical examination and thorax radiography were normal.

Material and methods

  1. Top of page
  2. Abstract
  3. Case report
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Study design

The patient had been away from the workplace for the 2 months previous to the study. The first day, he underwent clinical and physical evaluation and cutaneous tests, baseline methacholine-NSBH was assessed, and baseline blood and sputum samples were obtained. The following day, barley-BPT was performed and 2 weeks later L. destructor-BPT was carried out. Sputum samples were obtained 30 min and 18 h after both allergen challenges. Blood was sampled 18 h after each allergen-BPT, and methacholine-NSBH was assessed 24 h after each allergen-BPT. Every methacholine challenge test was carried out at least 6 h after sputum induction.

Skin tests

Cutaneous tests took the form of skin prick tests, as previously described (5). We tested a commercially available battery of allergens, including mites –Dermatophagoides pteronyssinus, D. farinae, L. destructor, and Tyrophagus putrescentiae at 100 BU/ml (Abelló, Spain), and Acarus siro, Blomia tropicalis, Euroglyphus maynei, and Gohiera fusca at 5000 E/ml (Aristegui, Spain) – flours – wheat, barley, rye, malt, soybean, oat, corn, at 5% w/v (Abelló, Spain) – pollens –Poa pratensis and Phragmites comunis at 100 BU/ml (Abelló, Spain) – molds –Alternaria tenuis at 100 BU/ml (Abelló, Spain) and Aspergillus fumigatus and Cladosporium herbarum at 5 w/v (Abelló, Spain) – and α-amylase at 1 mg/ml (CBF Leti, Spain). We also tested a sample of the barley dust and grain, provided by the patient, as previously described (5). Histamine phosphate at 10 mg/ml and PBS were used as positive and negative controls, respectively. The results of the skin tests were read at 15 min. Wheal diameters equal to or higher than 3 mm were considered positive in the absence of a response to PBS.

Bronchial provocation tests

NSBH was assessed with methacholine (Provocholine, Roche Laboratory, Nutley, NJ, USA) as agonist. Biologically standardized extracts of L. destructor and barley flour (Abelló SA, Madrid, Spain) were used in allergen-BPT. Agonist or allergen dilutions were administered with a MEFAR dosimeter (MEFAR s.r.l. Borezzo [BS], Italy), which was programmed to deliver five inhalations of 1 s each; the dosimeter administered 10 μl of solution in each inhalation. Tests were made after withholding inhaled short-acting β-adrenergic agonist for at least 6 h. A forced expiratory volume in 1 s (FEV1) higher than 70% predicted normal was required to start both tests. FEV1 values at basal stage and 3 min after diluent (PBS) inhalation were measured. A variability rate lower than 5% among basal and postdiluent FEV1 values was required to start the test.

Methacholine inhalation test

Methacholine dilutions at 0.125, 0.25, 0.5, 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 100.0, and 200.0 mg/ml with PBS as diluent were made. An agonist at increasing concentrations was administered with the dosimeter. FEV1 was measured 3 min after each inhalation. The test finished when a fall in FEV1 values equal to or higher than 20% from the postdiluent value was achieved, or when the highest concentration of methacholine was inhaled. The methacholine test was done at baseline and 24 h after each allergen-BPT. Results were expressed in terms of the provocative cumulative dose (given in μmol; 1 mol methacholine chloride=195.4 g) needed to decrease FEV1 by 20% of the baseline values (PD20M).

Allergen bronchial provocation test (BPT)

Allergen-BPTs were done first with barley extract at 5 w/v (Abelló, Spain), and 2 weeks later with Ld extract at 100 BU/ml (Abelló, Spain). Basal peak expiratory flow (PEF) was measured by means of a Mini-Wright peak flow meter (Clement Clark International Ltd, London, UK) before starting the test. Skin prick tests with twofold dilutions of allergen extract were performed to enable selection of a safe initial dose of allergen (concentration that produces a 3×3 mm wheal). As the barley extract cutaneous test was negative, we administered the highest extract concentration. Allergen was inhaled every 10 min with a twofold increasing allergen concentration at each step (FEV1 was measured at 10 min after each inhalation), until the highest dose of allergen was attained or there was an early asthmatic reaction. This was defined as a fall in FEV1 values equal to or higher than 20% from the postdiluent value. When the last dose of allergen was inhaled, FEV1 was recorded at 20, 30, 60, and 90 min. To record any late asthmatic reaction, PEF measurements were made hourly until 12 h after the challenge. A late asthmatic reaction was defined as a fall of 25% or more from the basal value (6).

Sputum induction

Sputum samples were obtained by means of hypertonic saline inhalation, as described by Fahy et al. (4, 7), at baseline and 30 min and 18 h after each allergen-BPT. Before sputum induction and to avoid contamination of the sample, subjects were asked to clean their mouth and nose. Four puffs of salbutamol were administered 30 min before sputum induction. With the aim of not interfering in the occurrence of the late asthmatic response (LAR), we did not administer salbutamol before the sputum induction carried out 30 min after allergen challenge. The PEF rate was recorded immediately before and after sputum induction. Saline at 5% was administered by an ultrasonic nebulizer model Ultraneb 99 (DeVilbiss, Somerset PA, USA), for 3 periods of 10 min each; after each period, the patient was asked to cough and expectorate into a sterile container. The test finished when a macroscopically adequate sputum sample was obtained or when the three periods of inhalation were completed.

The volume of the whole sputum sample was then recorded and mixed with an equal volume of Dithiotreitol (Sputasol, Unipath Ltd, Basingstoke, UK) at 1/100, and rocked at room temperature for 15 min. Then the mixture was filtered through one 0.42-μm Millipore filter (Millipore, Somerset, PA, USA) and centrifuged at 1500 g for 10 min. The supernatant was then aliquoted and frozen at −70°C until further analysis.

The pellet was suspended in saline at 0.9%, and standard cytologic stains (Papanicolau and Giemsa) were immediately made. Sputum samples were considered adequate for analysis when macrophages were visualized and squamous cell contamination was lower than 20% (3, 5). Percentual counts of macrophages, eosinophils, neutrophils, mast cells, lymphocytes, and epithelial cells were made over a total count of 400 cells.

In vitro tests

IgE measurements

Total serum IgE and specific IgE to the allergens which were positive in skin tests were measured by the Pharmacia CAP System IgE fluoro-enzyme immunosorbent assay (Pharmacia Diagnostics, Uppsala, Sweden).

Blood eosinophils and serum ECP

Total eosinophil counts and serum ECP levels were measured at baseline and 18 h after barley- and Ld-BPT.

ECP and tryptase level measurement

Serum ECP, and sputum supernatant ECP and tryptase levels were measured in duplicate and in the same assay by fluoro-enzyme immunosorbent assay (Pharmacia Diagnostics, Uppsala, Sweden).

Results

  1. Top of page
  2. Abstract
  3. Case report
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Cutaneous tests

We obtained positive results with Ld and with the barley dust provided by the patient (higher wheal diameters were 9 and 5 mm, respectively). The histamine control wheal size was 4 mm. All the other allergens tested, including those of the other mites, all the cereal flours, and the barley grain provided by the patient, were negative.

Blood measurements

Total blood eosinophils and serum ECP levels were, respectively, 559 cells/mm3 and 13.65 μg/l at baseline, 534 cells/mm3 and 15.48 μg/l 18 h after barley-BPT, and 639 cells/mm3 and 20.05 μg/l 18 h after Ld-BPT. Serum total IgE was 254 kU/l, and Ld-specific IgE was 2.1 kU/l. Barley- and D. pteronyssinus-specific IgE were negative.

Bronchial provocation tests

Basal spirometry was normal and barley-BPT was negative; Ld-BPT induced an isolated early bronchial response: the Ld-PD20 value was 20.08 BU/ml (FEV1 fall was 22%). Maximal PEFR fall was 17%, 7 h after Ld-BPT. The PD20M value decreased 24 h after Ld-BPT, but not after barley-BPT (Fig. 1).

image

Figure 1. Values of PD20M at baseline and 24 h after barley (B-BPT) and 24 h after L. destructor (Ld-BPT).

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Induced sputum

Sputum induction was safe, even during the early asthmatic response (PEF falls were always lower than 10%), and all the samples were adequate for cell counts (alveolar macrophages were visualized, and squamous cell contamination was under 20%). Sputum cell and chemicals results are shown in Table 1. Sputum eosinophil and epithelial cell percentages and ECP levels were increased (three-, nine- and fivefold from baseline values, respectively) 24 h after Ld-BPT. The highest tryptase levels were found 30 min (fourfold from basal values) after Ld-BPT. Barley-BPT did not modify sputum cell counts or chemical values.

Table 1.  Rate of inflammatory cells and chemicals in induced sputum
 Baseline30 min post-B18 h post-B30 min post-Ld18 h post-Ld
Epithelial cells (%)2.351.91.51.821.5
Eosinophils (%)5.46.17.135.7417.32
Macrophages (%)17.518.117.526.020.14
Neutrophils (%)73.472.771.163.138.29
Lymphocytes (%)0.731.12.252.82.5
ECP (μg/l)67.2102.498.7199.0385.0
Tryptase (μg/l)1.82.11.97.64.4

Discussion

  1. Top of page
  2. Abstract
  3. Case report
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Cereal workers are exposed to a wide diversity of substances with immunogenic capacity such as storage mites, molds, pollens, and amylase, which can contaminate cereal dust, and to which, they can become sensitized (8). We report the case of a miller exhibiting hypersensitivity to the storage mite L. destructor without cereal allergy.

Today, bronchial provocation tests are considered the “gold standard” in the diagnosis and assessment of OA (1, 2). Two bronchial response patterns can be elicited by allergen-BPT. The early asthmatic reaction (EAR) starts 10–20 min after BPT and lasts 1–3 h, while the late asthmatic response (LAR) starts 3 h after BPT, and may increase NSBH for several weeks. Current evidence strongly suggests that the EAR is due to the IgE-dependent release of mediators, mainly from mast cells, while airway eosinophilic inflammation and activation constitute the underlying mechanism involving the LAR. Bronchoalveolar lavage (BAL) and bronchial biopsy have demonstrated their accuracy in the study of the inflammatory events occurring within the airways after allergen challenge. Nevertheless, these techniques do not lack risk (10), are expensive, and cause too much patient annoyance to be routinely considered in the assessment of OA.

In order to study the inflammatory changes induced by allergen inhalation during both the EAR and the LAR, we sampled sputum 30 min and 18 h after Ld-BPT. To evaluate the specificity of the changes, we challenged the patient with barley, an allergen to which he was exposed but not sensitized. Although mast cells are probably involved in the development of EAR, we did not identify them in sputum samples, even in those obtained 30 min after Ld-BPT. We are aware that this lack of detection might be due to the fact that we did not use specific mast-cell staining (toluidine blue), that the number of cells counted in each preparation (400 cells) might be low, or even that the high centrifugation speed might have broken such cells. However, other authors have also reported difficult mast-cell detection both in BAL (11) and in sputum (3, 12), a fact what might suggest that mast cells are usually placed at the mucous layer and do not migrate into the airway lumen. Tryptase is a selective marker of mast-cell activation, but its quantification in the airway fluid has been controversial, since some authors have reported higher tryptase levels in asthma patients than controls (13), but others have not detected such differences (7). Thirty minutes after Ld-BPT, we found an increase in tryptase level (fourfold) from baseline. Sputum tryptase values, even when higher than baseline, decreased 18 h after Ld-BPT to the EAR level. Our results agree with other authors who have reported an enhancement of BAL (14) and sputum (15) tryptase levels 12 min and 4 and 24 h after allergen-BPT, levels that tend to become normal 48 h after allergen-BPT (14).

The LAR after allergen challenge is characteristically eosinophilic (16), and BAL samples obtained at different times during the LAR demonstrate an increase of eosinophils after exposure to occupational allergens such as plicatic acid (17). Although we did not identify a clear LAR in our patient, eosinophil percentages and ECP levels were clearly increased in the sputum obtained 18 h after Ld-BPT, but not after barley-BPT, when compared to the baseline values. Sputum eosinophil numbers and ECP levels in our study were lower than reported in the literature (4, 7, 13), a finding which could be attributed to the dilution of the whole sputum sample by saliva (13). However, our results are also lower than those reported by other authors also analyzing the entire sputum sample, but studying more severe asthma (4,7). Thus, we think that the lower eosinophilic inflammation markers found in our study are dependent on the sputum sample dilution but also on the mildness of the disease. ECP is an eosinophilic activation marker that can damage respiratory epithelium (18). In our study, it showed an increase in epithelial cell numbers in the sputum sample obtained 18 h after Ld-BPT. Since epithelial damage can increase the airway permeability to the agonist, the higher sputum epithelial cell percentages are consistent with the increase in NSBH found in our study 24 h after Ld-BPT.

To our knowledge, only Maestrelli et al. (12) have used induced sputum to evaluate the inflammatory changes induced by occupational agents-BPT in the airway. They determined the eosinophil level in sputum plugs 8 and 24 h after allergen-BPT, and reported an increment in such cells which was independent of the bronchial response type provoked by the allergen, but they did not evaluate the immunologic response during the EAR, nor measure chemicals in sputum. To our knowledge, this is the first study in which induced sputum has been used to evaluate the airways immunologic response during both the EAR and the LAR induced by occupational allergen-BPT.

The patient presented in this report suffered from mild asthma. Lung-function tests were nearly normal, and only a mild isolated immediate response was obtained after allergen-BPT. However, we found an increase in sputum tryptase levels during the EAR, as well as an increase in the number of eosinophils and epithelial cells and in the levels of ECP in sputum, 18 h after Ld-BPT, a finding which is consistent with the increase in NSBH. Since airway inflammation is the central feature of asthma, our results suggest that the assessment of the inflammatory airway component provides useful supplementary information to the lung-function tests in the management of OA. The role of the induced sputum technique in asthma inflammation study has been widely standardized in recent years (4). Furthermore, it is a noninvasive as well as an easy, fast, and cheap method, indicating that its use in monitoring asthma is more feasible than BAL sampling. Thus, we think that induced sputum analysis might be routinely applied to diagnose and monitor OA.

References

  1. Top of page
  2. Abstract
  3. Case report
  4. Material and methods
  5. Results
  6. Discussion
  7. References