M.J. AlvarezR. CastilloA. ReyN. OrtegaC. BlancoT. Carrillo

Authors' affiliations:

M.J. Alvarez, R. Castillo, N. Ortega, C. Blanco,

T. Carrillo, Allergy Department, Hospital

Universitario Nuestra SenÄ ora del Pino, Las

Palmas de Gran Canaria

A. Rey, Pathology Department, Hospital

Universitario Nuestra SenÄ ora del Pino, Las

Palmas de Gran Canaria, Spain

Correspondence to:

MarõÂa J. Alvarez Puebla

Calle Juan XXIII, n81,38D1

35004 Las Palmas GC

Spain

Date:

Accepted for publication 6 April 1999

To cite this article:

Alvarez M.J., Castillo R., Rey A., Ortega N., Blanco C. &

Carrillo T. Occupational asthma in a grain worker due

to Lepidoglyphus destructor, assessed by bronchial

provocation test and induced sputum.

Allergy 1999, 54, 884±889.

Copyright # Munksgaard 1999

ISSN 0105-4538

Case report

Occupational asthma in agrain worker due toLepidoglyphus destructor,assessed by bronchial

provocation test and inducedsputum

Key words: airway inflammation; ECP; induced sputum;

occupational asthma; tryptase.

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 non-

invasive 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 methacho-

line 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.

884

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 demon-

strated 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

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

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, base-

line 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 (Abello , 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 (Abello , Spain) ± pollens

± Poa pratensis and Phragmites comunis at 100 BU/ml

(Abello , Spain) ± molds ± Alternaria tenuis at 100 BU/ml

(Abello , Spain) and Aspergillus fumigatus and

Cladosporium herbarum at 5 w/v (Abello , Spain) ± and a-

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 (Abello SA, Madrid, Spain) were used in

allergen-BPT. Agonist or allergen dilutions were adminis-

tered 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 ml of solution in

Alvarez et al . Asthma caused by L. destructor

Allergy 54, / 884±889 | 885

each inhalation. Tests were made after withholding inhaled

short-acting b-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 admin-

istered 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 methacho-

line 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 mmol; 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

(Abello , Spain), and 2 weeks later with Ld extract at 100 BU/

ml (Abello , 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

333 mm wheal). As the barley extract cutaneous test was

negative, we administered the highest extract concentra-

tion. 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-mm Millipore filter (Millipore,

Somerset, PA, USA) and centrifuged at 1500 g for 10 min.

The supernatant was then aliquoted and frozen at ±708C

until further analysis.

The pellet was suspended in saline at 0.9%, and standard

cytologic stains (Papanicolau and Giemsa) were immedi-

ately 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.

Alvarez et al . Asthma caused by L. destructor

886 | Allergy 54, / 884±889

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

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 mg/l at baseline, 534

cells/mm3 and 15.48 mg/l 18 h after barley-BPT, and 639

cells/mm3 and 20.05 mg/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).

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.

Discussion

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.

Ld P

D20m

ethach

oline

(µm

ol)

1·6

1·5

1·4

1·3

1·2

1·1

1·0

0·9

Baseline 24 h afterB-BPT

24 h afterLd-BPT

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

24 h after L. destructor (Ld-BPT).

Table 1. Rate of inflammatory cells and chemicals in induced sputum

Baseline 30 min post-B 18 h post-B 30 min post-Ld 18 h post-Ld

Epithelial cells (%) 2.35 1.9 1.5 1.8 21.5

Eosinophils (%) 5.4 6.1 7.13 5.74 17.32

Macrophages (%) 17.5 18.1 17.5 26.0 20.14

Neutrophils (%) 73.4 72.7 71.1 63.1 38.29

Lymphocytes (%) 0.73 1.1 2.25 2.8 2.5

ECP (mg/l) 67.2 102.4 98.7 199.0 385.0

Tryptase (mg/l) 1.8 2.1 1.9 7.6 4.4

Alvarez et al . Asthma caused by L. destructor

Allergy 54, / 884±889 | 887

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 inflam-

mation 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 tech-

niques 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 eosino-

phils 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

Alvarez et al . Asthma caused by L. destructor

888 | Allergy 54, / 884±889

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.

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