Fluticasone and ibuprofen do not add to the effect of salmeterol on organic dust-induced airway...

doi: 10.1111/j.1365-2796.2008.01928.x

Fluticasone and ibuprofen do not add to the effect ofsalmeterol on organic dust-induced airway inflammationand bronchial hyper-responsiveness

K. Strandberg, A. Ek, L. Palmberg & K. Larsson

From the Lung and Allergy Research, The National Institute of Environmental Medicine, Karolinska Institutet, Stockholm,Sweden

Abstract. Strandberg K, Ek A, Palmberg L, Larsson K.

(Karolinska Institutet, Stockholm, Sweden). Flutica-

sone and ibuprofen do not add to the effect of salme-

terol on organic dust-induced airway inflammation

and bronchial hyper-responsiveness. J Intern Med2008; 264: 83–94.

Background. Exposure in a pig house causes airway

inflammation and bronchial hyper-responsiveness

which are not influenced by anti-asthma drugs, includ-

ing a b2-agonist (salmeterol).

Objectives. We hypothesized that a glucocorticoid or a

cyclo-oxygenase-inhibitor synergistically interacts

with salmeterol offering a protection against dust-

induced increased bronchial responsiveness and air-

way inflammation. As data did not confirm previous

results a retrospective analysis of pooled data on dust-

induced bronchial hyper-responsiveness from four

other studies was performed.

Design. Fluticasone or ibuprofen was administered for

1 week and salmeterol or placebo was inhaled 1 h

prior to a 3-h exposure in a pig barn in a double-

blind, placebo-controlled, cross-over design

(2–3 weeks apart) in 12 healthy subjects. Lung func-

tion, bronchial responsiveness to methacholine and

inflammatory markers were evaluated before and after

exposure. Pre- and postexposure bronchial responsive-

ness in nontreated subjects was retrospectively evalu-

ated from four previous studies.

Subjects. Twelve healthy, nonatopic nonsmokers.

Results. Salmeterol partially protected against bron-

chial hyper-responsiveness but did not influence

inflammatory markers. Fluticasone and ibuprofen did

not add to these effects. The retrospective analysis

showed that PD20FEV1 after exposure in a pig barn is

almost totally independent of pre-exposure

PD20FEV1-level; all subjects end up at the same low

postexposure PD20FEV1.

Conclusion. Contradictory to our previous results,

salmeterol offered partial protection against enhanced

bronchial responsiveness induced by exposure in a

pig barn. This effect was not modified by fluticasone

or ibuprofen. Our data clearly demonstrate that inter-

ventions altering bronchial responsiveness must be

compared between groups with similar prechallenge

bronchial responsiveness or in a cross-over design.

Keywords: cyclo-oxygenase-inhibitor, glucocorticoster-

oid, lung function, methacholine provocation, b2-ago-

nist.

ª 2008 Blackwell Publishing Ltd 83

Original Article |

Introduction

Exposure of healthy subjects in swine confinement

buildings induces an intense airway inflammation and

increased bronchial responsiveness to direct stimuli

[1–5]. We have previously shown that the long-acting

b2-agonist salmeterol does not protect against the

increased bronchial responsiveness induced by expo-

sure in a pig barn in healthy subjects [6]. This some-

what surprising lack of protection is unlikely to be

explained by tachyphylaxis as exposure in the pig

barn induced a similar enhancement of bronchial

responsiveness after one dose and after 2 weeks of

regular dosing of salmeterol. It has also been demon-

strated that 2 weeks of inhalation of a glucocorticoid

(fluticasone) prior to exposure in a pig house does not

influence postexposure increase of bronchial respon-

siveness [1].

Inflammatory cytokines, such as interleukin (IL)-1band tumour necrosis factor (TNF), attenuate the ability

of cultured airway smooth muscle cells to relax in

response to b-adrenoceptor agonists, an effect that has

been claimed to involve cyclo-oxygenase (COX)-2 [7,

8]. According to this hypothesis COX-2 increases

PGE2 release, resulting in increased cyclic adenosine

monophosphate (cAMP) formation which in turn

leads to PKA activation and thereby phosphorylation

and desensitization of the b-adrenoceptor [8]. As IL-1

and TNF are involved in the airway inflammatory

reaction following exposure in a pig barn [3, 9, 10],

we assumed that the previously described lack of pro-

tective effect of salmeterol against dust-induced bron-

chial hyper-responsiveness [6] may be caused by

heterologous desensitization of airway b2-adrenocep-tors induced by pro-inflammatory cytokines. If so,

inhibition of COX-2 would enhance b2-adrenoceptor

function leading to protection of a b2-agonist againstenhanced bronchial responsiveness induced by expo-

sure in a pig house.

In the treatment of asthma a combination of b2-adre-noceptor agonists and glucocorticoids is more effec-

tive than either drug alone. Glucocorticoids may

enhance b2-adrenoceptor function by increasing

mRNA expression of the b2-adrenoceptor protein [11],

and b2-agonists in turn may facilitate glucocorticoid

receptor (GR) nuclear localization and enhance GR

binding to its specific target DNA sequences [11, 12].

In the present study the aim was to examine whether

a glucocorticosteroid (fluticasone) or a COX-inhibitor

(ibuprofen) influences the protective effect of salme-

terol on the increased bronchial responsiveness and

the acute inflammatory response in healthy subjects

after exposure in a pig barn. In our previous, parallel

group study [6] we could not exclude the possibility

that the study design may have influenced the results

and thereby the interpretation of data. By performing

the present cross-over study possible influence of

inter-individual differences in preexposure bronchial

responsiveness is eliminated.

When we found that the results of the present study

did not confirm our previous finding, i.e. a lack of

protection of salmeterol against exposure-induced

increase in bronchial responsiveness, we conducted a

retrospective analysis of pooled data from four previ-

ous studies of bronchial responsiveness before and

after exposure in a pig barn in healthy subjects. The

aim of this separate, retrospective, analysis was to

improve our understanding on bronchial hyper-respon-

siveness induced by exposure in a pig barn in order

to better understand the discrepancy between the pres-

ent and previous results.

Materials and methods

Subjects

Twelve healthy, nonatopic nonsmokers with no his-

tory of allergic diseases, asthma or other airways dis-

eases participated in the study. Atopy was evaluated

by a questionnaire and a skin-prick test with extracts

from 17 common aeroallergens. Normal results of

physical examination, spirometry and bronchial

responsiveness were required for inclusion. According

to our own reference values for bronchial responsive-

ness to methacholine, a PD20FEV1 >0.26 mg for

women (n = 101) and >0.56 mg for men (n = 102) is

regarded as normal (defined as the 10th percentile).

For subject’s characterization see Table 1. The study

K. Strandberg et al. | Salmeterol and bronchial response

84 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 264; 83–94

was approved by the ethics committee at Karolinska

Institutet and all subjects gave informed consent.

Study design

Twelve subjects were, in a randomized (randomization

according to blocks of four), double-blind, cross-over

design, treated for 1 week either with fluticasone or

placebo inhalations and ibuprofen or placebo tablets

with a wash out period of 2–3 weeks between the

treatment periods (study design is shown in Table 2).

In the morning, 1 h prior to exposure in the pig barn,

either salmeterol or placebo was inhaled. The expo-

sure took place in a pig barn, containing 300–400

pigs, during weighing of pigs for 3 h.

Symptoms were assessed 1.5 h before and approxi-

mately 8 h after the start of the exposure. Spirometry,

a bronchial methacholine challenge and measurement

of exhaled nitric oxide (NO) were performed 2 weeks

before the first treatment period and 7 h after the start

of exposure in the pig barn following all four treat-

ment periods. Induced sputum and blood samples

were collected 2 weeks prior to the first exposure and

8 and 6.5 h, respectively, after the start of each expo-

sure. Peak expiratory flow (PEF) was measured on

the exposure day before medication, 1 h after medica-

tion, i.e. immediately prior to exposure and at 0, 1, 2,

3 and 4 h after cessation of exposure.

Exposure measurements

Inhalable dust (<10 lm; IOM inhalable dust sampler,

SKC Ltd, Dorset, UK) and respirable dust (<5 lm;

plastic cyclone samplers, Casella Ltd, London, UK)

were sampled, weighed and analysed for endotoxin

by the use of a kinetic technique version of Limulusamebocyte lysate assay (Limulus Amebocyte lysate;

Endosafe� Endochrome-KTM, Coatech AB, Kungsba-

cka, Sweden), with Escherichia. coli 0111:B4 as stan-

dard.

Symptoms

The subjects indicated five general and seven airway

specific symptoms on a 100 mm visual analogue scale

(VAS) before and 7 h after exposure in accordance

with previous studies [13–15]. The subjects were

requested to put a cross on the scale where 0 indi-

cated no and 100 unbearable symptoms. Oral temper-

ature was measured directly before exposure and then

every hour for 8 h.

Lung function

Spirometry (forced expiratory volume in 1 s, FEV1

and vital capacity, VC) was measured using a wedge

spirometer (Vitalograph�; Medical Instrumentation,

Buckingham, UK) according to recommendations of

Table 1 Subjects characteristics

Men ⁄women 5 ⁄ 7Age, years, mean (range) 30 (21–54)

FEV1, % of predicted, mean (SD) 98 (8.8)

VC, % of predicted, mean (SD) 95 (8.1)

FEV1 ⁄VC, %, mean (SD) 82 (7)

PD20FEV1, mg, median (25th–75th percentiles) 1.86 (0.56–7.2)

Table 2 Treatment regimens

Treatment during 1 week prior to exposure

A single dose, inhaled 1 h prior

to exposure

Inhalation Oral intake Inhalation

Fluticasone Placebo Ibuprofen Placebo Salmeterol, 50 lg Placebo

Placebo ⁄ placebo 2 inh b. i. d. 1 tabl b. i. d. 2 inh

Placebo ⁄ salmeterol 2 inh b. i. d. 1 tabl b. i. d. 2 inh

Fluticasone ⁄ salmeterol 500 lg b i d 1 tabl b. i. d. 2 inh

Ibuprofen ⁄ salmeterol 2 inh b. i. d. 600 mg b i d 2 inh


ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 264; 83–94 85

the American Thoracic Society [16]. Local reference

values were used [17, 18]. PEF was measured with a

mini-Wright peak flow meter (Clement Clarke Ltd,

London, UK). The best of three blows was registered.

Methacholine provocation

After inhalation of the diluent, increasing concentra-

tions of methacholine was inhaled, starting at

0.5 mg mL)1 with doubling of the concentration up

to 64 mg mL)1 or until FEV1 decreased by 20% [19].

With our bronchial challenge method we have previ-

ously shown that PD20FEV1 could be defined in 80%

of healthy subjects [19, 20]. The results were

expressed as the cumulative dose causing a 20%

decrease in FEV1 (PD20FEV1). In one subject the

FEV1-decrease was <20% (14%) at the highest meth-

acholine concentration (64 mg mL)1), corresponding

to a PD20FEV1 of 38 mg, before exposure. In this

subject an extrapolated preexposure PC20FEV1 value

was used for statistical calculations.

Exhaled NO

Exhaled nitric oxide (NO) was analysed with chemilu-

minescence after reaction with ozone (NIOX�; Aero-

crine, Stockholm, Sweden) and was assessed

according to the recommendations of the European

Respiratory Society [21] and the American Thoracic

Society [22]. To minimize contamination from the

oral cavity, mouth-wash with water and sodium bicar-

bonate (10%) (half a minute each), preceded the mea-

surement procedure [23].

Blood samples

Blood samples were allowed to coagulate at room

temperature for 1 h before centrifugation at 1000 gfor 10 min. Serum was dispensed in aliquots kept at

)70 �C until analysis. Each sample underwent only

one freeze-thaw cycle before assay.

Sputum

Sputum induction was performed according to in ‘t

Veen et al. [24] with minor modifications. After inha-

lation of 400 lg salbutamol, sputum was induced by

inhalation of increasing concentrations of saline using

an ultrasonic nebulizer (De Vilbiss Ultraneb 2000; De

Vilbiss Healthcare Worldwide, Somerset, PA) with an

output of 3 mL min)1. The concentrations of saline

were 0.9%, 3%, 4% and 5% and each concentration

was inhaled for 7 min followed by measurement of

FEV1. After each concentration the subjects blew the

nose and rinsed the mouth with water, and were asked

to cough deeply and expectorate sputum.

The sputum sample was considered adequate when it

macroscopically appeared to be free from saliva and

weighing at least 1000 mg. The colour and the weight

of the entire sputum were determined. An equal vol-

ume of (dithiothreitol) DTT 0.1% was added to the

sample for a final concentration of 0.05% DTT and

rocked for 15–25 min in a 37 �C waterbath for

homogenization and dissociation of disulphide bonds.

The sample was filtered and centrifuged for 10 min at

400 g, and the supernatant was dispensed into several

aliquots, which were kept in )70 �C until analysis.

The cell pellet was resuspended in 2 mL PBS and a

total cell count and viability test with Trypan blue

was performed. The cell suspension was adjusted for

cytospin, and a differential cell count was performed.

Cytokine analyses

Analyses of IL-6 in blood and IL-6, IL-8 and TNF in

sputum were performed. The IL-6 and IL-8 concentra-

tions were determined by ELISA methods developed in

our laboratory using commercially available antibody

pairs, for details see [25]. Controls at three different

concentrations were used as calibrators in both methods

and cytokine concentration was expressed as pg mL )1.

For duplicates an intra- and inter-assay coefficient of

variation of <10% and 20% respectively, was accepted.

The detection ranges for IL-6 was 3–375 pg mL )1 and

for IL-8 50–3200 pg mL )1. TNF was analysed using

commercial high sensitive sandwich enzyme immuno-

assay kits (QuantikineTM R&D Systems, Europe Ltd,

Abingdon, UK). Absorbance was read at 490 and

650 nm with a Thermomax 250 reader (Molecular

Devices; Sunnyvale, CA). The detection range was

0.5–32 pg mL )1.



Retrospective analysis of bronchial responsiveness

Data from previous studies [1, 26, 27] and one yet

unpublished study in which pre- and postexposure

bronchial responsiveness to methacholine has been

assessed were pooled and analysed. Data were

obtained from 47 healthy nonsmokers who underwent

a bronchial methacholine challenge before and after

exposure in a pig house, identical with the procedure

of the present study. The 47 subjects included in the

retrospective analysis were not treated with any active

drug and were not using masks or other protective

devices during exposure.

Statistics

Normal distribution of the pre-exposure data was

confirmed by Z-score. Data on lung function, exhaled

NO, body temperature and symptoms are presented

as mean ± SEM or range, and comparisons were

made using analysis of variance (anova). Because

of the cross-over design, all comparisons are calcu-

lated as dependent observations which do not allow

traditional posthoc tests. Therefore, Student’s t-testfor paired observations was used as posthoc test

when the outcome of the anova was found signifi-

cant (P < 0.05). Data on bronchial responsiveness,

serum, sputum and concentrations of airborne dust

and endotoxin are presented as median values (25th–

75th percentiles), and comparisons were made using

anova and Student’s t test for paired observations

on log10-transformed data. A P-value <0.05 was con-

sidered significant. Cytokine concentration values

below the detection limit were assigned a value of

1.5 pg mL)1 for IL-6, 30 pg mL)1 for IL-8 and

0.3 pg mL)1 for TNF. One subject did not attend

one of the exposures and is therefore excluded from

the between-treatment comparisons. Statistical analy-

sis was performed using software (StatView, ver-

sion 5.0.1; SAS Institute; Cary, NC, USA). In the

retrospective analyses comparisons are made by

means of simple regression on absolute numbers or

log2-transformed data.

Results

Exposure measurements

Exposure to airborne dust and endotoxin was similar

at the four exposure occasions (Table 3).

Body temperature and symptoms

The maximal increase of postexposure body tempera-

ture did not differ between treatments (mean increase

0.70–0.86 �C, range 0.0–2.6 �C). There was a differ-

ence in exposure-induced symptoms between the dif-

ferent periods (F = 3.00, P = 0.044) and, in general,

exposure induced most symptoms during the flutica-

sone ⁄ salmeterol treatment period. Cough was the

most prominent symptom irrespective of treatment.

Pre- and postexposure changes of symptoms are given

in Table 4.

Lung function

Exposure in the pig house caused a slight but sig-

nificant impairment of FEV1 and VC following

placebo ⁄placebo treatment (Table 5). The fall in

FEV1 and VC tended to be more pronounced after

Table 3 Expoure to airborne endotoxin and dust during exposure in a pig barn

Placebo ⁄placebo

Placebo ⁄salmeterol

Fluticasone ⁄salmeterol

Ibuprofen ⁄salmeterol

Difference between

groups

Inhalable dust (mg m)3) 24.7 ± 3.64 22.6 ± 3.84 25.3 ± 4.13 22.3 ± 4.03 F = 0.16; P = 0.92

Inhalable endotoxin (ng m)3) 189 ± 17.5 167 ± 13.9 193 ± 15.9 176 ± 15.2 F = 1.24; P = 0.40

Respirable dust (mg m)3) 0.432 ± 0.028 0.396 ± 0.027 0.448 ± 0.024 0.411 ± 0.026 F = 0.72; P = 0.55

Respirable endotoxin (ng m)3) 7.10 ± 0.56 7.32 ± 0.86 8.47 ± 0.67 7.51 ± 0.76 F = 0.86; P = 0.47

Values are given as mean ± SEM



fluticasone ⁄ salmeterol than in placebo ⁄ salmeterol and

ibuprofen ⁄ salmeterol (Table 5).

A significant decrease in PEF was found after pla-

cebo ⁄placebo treatment only, and postexposure PEF

was significantly lower after placebo ⁄placebo than

after the other treatments (Fig. 1). The maximal post-

exposure PEF reduction (compared with postmedica-

tion, preexposure values) was 49 ± 6.5 L min)1 after

placebo ⁄placebo, 31 ± 9.8 L min)1 after placebo ⁄salmeterol, 37 ± 16 L min)1 after fluticasone ⁄ salme-

terol and 15 ± 10 L min)1 after ibuprofen ⁄ salmeterol

treatment (Fig. 1).

Bronchial responsiveness

Exposure in the pig barn induced a significant

enhancement of bronchial responsiveness to methach-

oline after placebo ⁄placebo, fluticasone ⁄ salmeterol

and ibuprofen ⁄ salmeterol but not after placebo ⁄ salme-

terol when compared with pre-exposure values

(Fig. 2a). The increase in bronchial responsiveness

was 4.0 (2.2–5.0) doubling dose steps after pla-

cebo ⁄placebo, 0.88 (0.22–1.9) after placebo ⁄ salmeter-

ol, 1.9 (0.21–2.7) after fluticasone ⁄ salmeterol and 2.1

(1.1–2.5) after ibuprofen ⁄ salmeterol treatment. Com-

pared with placebo ⁄placebo there was a significant

Table 4 Difference in symptoms after–before exposure inmm on VAS.

Symptom Placebo Salmeterol

Fluticasone ⁄

salmeterol

Ibuprofen ⁄

salmeterol

Chills 4.1 ± 2.8 6.9 ± 5.8 19 ± 5.9 10 ± 5.8

Headache 2.3 ± 2.8 9.3 ± 3.7 12 ± 6.5 7.2 ± 3.5

Fatigue 12 ± 5.7 17 ± 6.8 7.1 ± 3.7 11 ± 6.2

Muscle pain 6.4 ± 2.4 5.3 ± 3.1 5.4 ± 3.0 4.3 ± 2.8

Nausea )1.8 ± 1.9 2.1 ± 2.0 5.3 ± 4.1 4.4 ± 2.4

Sneezing 3.6 ± 2.1 5.5 ± 3.1 0.4 ± 4.0 4.3 ± 3.2

Nasal congestion 6.6 ± 5.3 7.6 ± 3.2 14 ± 5.8 5.7 ± 3.0

Nasal secretion )0.1 ± 2.6 )0.5 ± 3.3 2.1 ± 3.4 6.6 ± 4.3

Cough 23 ± 5.7 19 ± 6.0 25 ± 5.4 18 ± 4.9

Chest tightness 5.9 ± 2.4 4.5 ± 3.8 13 ± 5.3 3.3 ± 3.8

Dyspnea 2.8 ± 2.1 )0.1 ± 0.8 7.2 ± 4.0 1.1 ± 0.9

Wheezing 0.1 ± 0.08 0.5 ± 0.4 0.7 ± 0.3 0.3 ± 0.1

Sum 65 77 111 76

Values are given as mean ± SEM.

Table 5 FEV1 and VC 2 weeks before and 7 h after exposure in a pig barn.

Preexposure

No medication

(% of predicted value)

Postexposure Change in % of pre-exposure

Difference between

groups

Placebo ⁄placebo

Placebo ⁄salmeterol

Fluticasone ⁄salmeterol

Ibuprofen ⁄salmeterol

FEV1 97.9 ± (8.8) )6.6 ± 3.9*** )1.0 ± 3.5�� )3.6 ± 5.6*� )3.2 ± 6.2 F = 4.9 P = 0.003

VC 95.4 ± 8.1 )3.7 ± 4.4** )2.1 ± 3.1* )4.4 ± 4.4** „ )2.5 ± 4.9 F = 6.0 P = 0.0007

*P < 0.05, **P < 0.01, ***P < 0.001 indicate comparison with pre-exposure value.�P < 0.05, ��P < 0.001 indicate comparison with placebo ⁄ placebo.„ P < 0.05 indicates comparison with placebo ⁄ salmeterol.values are given as mean ± SEM.

Ibuprofen + salmeterolFluticasone + salmeterolPlacebo + salmeterolPlacebo + placebo

440

460

480

500

520

540

560

580

PE

F (

l min

–1)

–1 0 3 4 5 6 Hours

******

**

***

***

***

**** *

***

Pig houseexposure

Preexposure Post exposure

***

##

######

Fig. 1 Peak expiratory flow (PEF) before and after 3 hexposure in a pig barn. n = 12 except for placebo ⁄ salmeteroltreatment where n = 11. Mean and SEM. There was no sig-nificant difference between the three periods when salmeterolwas inhaled prior to exposure. *P < 0.05, **P < 0.01,***P < 0.001 indicate comparison with placebo ⁄ placebo.#P < 0.05, ###P < 0.001 indicate comparison with 1 h post-drug, pre-exposure value, i.e. at time point 0 h.



protection in all other three arms (Fig. 2). Postexpo-

sure PD20FEV1 did not differ between the three peri-

ods with preexposure salmeterol inhalations.

Exhaled NO

Pig house exposure caused increased levels of exhaled

NO (F = 3.00, P = 0.03) after placebo ⁄placebo(P = 0.005) and fluticasone ⁄ salmeterol (P = 0.035)

treatment (Fig. 2b). Placebo ⁄ salmeterol and ibupro-

fen ⁄ salmeterol treatment resulted in significantly

lower exhaled NO levels compared with placebo ⁄pla-cebo treatment (both P = 0.03) while flutica-

sone ⁄ salmeterol treatment did not differ significantly

from placebo ⁄placebo.

Analyses in serum and sputum

Postexposure IL-6 in serum increased after all periods

(F = 4.5, P = 0.0042) with no significant differences

between treatments (Fig. 2c).

The total number of cells (F = 13.0, P < 0.0001) and

the number of neutrophils (F = 29.7, P < 0.0001) in

sputum increased after exposure. Sputum cell count

did not differ between treatments (Fig. 3).

Exposure induced increase of IL-6 (F = 27.4,

P < 0.0001), IL-8 (F = 15.5, P < 0.0001) and TNF

(F = 3.31, P = 0.021) levels in sputum irrespective of

treatment with no significant differences between

treatments (Fig. 4).

Bronchial responsiveness (retrospective data)

There was a weak correlation between the postexpo-

sure increase in bronchial responsiveness calculated

as doubling of the PD20FEV1 and pre-exposure bron-

chial responsiveness (Fig. 5a) whereas the correlation

between pre-exposure PD20FEV1 and the pre- and

postexposure difference in PD20FEV1 was almost

perfect (Fig. 5b). This excellent correlation is

explained by the fact that postexposure bronchial

responsiveness ends up at a similar level in all sub-

jects irrespective of pre-exposure PD20FEV1-level

(Fig. 5c). There is, however, a correlation between

pre-and postexposure bronchial responsiveness and

those with a high preexposure PD20FEV1 is also

exhibiting a slightly higher PD20FEV1 after exposure

(Fig. 5d).

0.050.10.20.40.81.63.26.4

12.8(a)

(b)

PD

20 F

EV

1 (m

g)

###

Pre exposure

Placebo/placebo

Placebo/salmeterol

Fluticasone/salmeterol

Ibuprofen/ salmeterol

Post exposure

0

5

10

15

20

NO

(p

pb

)

#

Median and interquartile range

Mean and SEM

***

* *

#

######

***

(c)

0

10

20

30

40

50

IL-6

(p

g m

L–1

)

Preexposure

Placebo/placebo

Placebo/salmeterol

Fluticasone/ salmeterol


Post exposure

Median and interquartile range ***

***

Fig. 2 Bronchial responsiveness to methacholine, levels ofexhaled nitric oxide (NO) and IL-6 serum levels before andafter exposure in a pig barn. n = 12 except for pla-cebo ⁄ salmeterol treatment where n = 11. PD20FEV1 beforeexposure was 1.86 (0.56–7.16) mg. Pre-exposure level ofexhaled NO was 12.4 ± 1.60 ppb. Pre-exposure blood levelof IL-6 was 1.5 (1.5–11.6) pg mL)1. *P < 0.05,**P < 0.01, ***P < 0.001 indicate comparison with pre-exposure values. #P < 0.05, ###P < 0.001 indicate compari-son with placebo ⁄ placebo treatment.



Discussion

The main aim of the present study was to further

explore our previous, somewhat surprising, finding

that a long-acting b2-agonist (salmeterol) did not pro-

tect against enhanced bronchial methacholine respon-

siveness induced by exposure in a pig house [6]. We

were, however, not able to confirm our previous

results and in the present study we demonstrated that

pre-exposure inhalation of salmeterol offered a partial

protection against increased bronchial responsiveness

following exposure in a pig house. This protection

Mac

rop

hag

es m

g–1

sp

utu

m

0 250 500 750

1000 1250 1500 1750 2000

0

50

100

150

200

250

Lym

ph

ocy

tes

mg

–1 s

pu

tum

0

2000

4000

6000

8000

10000

(a)

(c)

(b)

(d)

To

tal n

um

ber

of

cells

N

eutr

op

hils

mg

–1 s

pu

tum

0

2000

4000

6000

8000

10000

**

***

*** ***

**

*** ***

***

Preexposure

Placebo/ placebo

Placebo/ salmeterol



Post exposurePre

exposure

Placebo/ placebo

Placebo/ salmeterol



Post exposure

mg

–1 s

pu

tum

Fig. 3 Cells in sputum before and after exposure in a pig barn. n = 11. Pre-exposure values (median and 25th–75th percen-tiles): Total cell number 804 (482–1119) cells mg)1 sputum, macrophages 515 (298–713) cells mg)1 sputum, neutrophils 78(44–379) cells mg)1 sputum and lymphocytes 14 (7.6–26) cells mg)1 sputum. No difference between treatments. *P < 0.05,**P < 0.01, ***P < 0.001 indicate comparison with pre-exposure values.

IL-6

(n

g m

l–1 )

0

0.5

1.0

1.5

2.0

2.5

3.0

***

***

*** ***

(a) (b)

0

2

4

6

8

10

12

14

16

TN

F (

pg

ml–

1 )

*

*****

Placebo/placebo

Placebo/salmeterol


Ibuprofen/salmeterol

Preexposure

Post exposure

Placebo/placebo

Placebo/salmeterol



Preexposure

Post exposure

0

0.5

1.0

1.5

2.0

2.5

3.0

IL-8

, (n

g m

l–1 )

***

********

(c)

Placebo/placebo

Placebo/salmeterol



Preexposure

Post exposure

Fig. 4 Cytokines in sputum before and after exposure in a pig barn. n = 12 for IL-6 and IL-8 except for the placebo ⁄ salmeter-ol period where n = 11. Pre-exposure values (median and 25th–75th percentiles): IL-6 28 (12–59) pg mL)1, IL-8 541 (302–821) pg mL)1, TNF 0.30 (0.30–1.6) pg mL)1. *P < 0.05, **P < 0.01, ***P < 0.001 indicate comparison with pre-exposurevalues.



was similar when salmeterol was given together with

placebo, fluticasone and ibuprofen. In addition, one

single dose of salmeterol, by itself or in combination

with fluticasone or ibuprofen treatment, did not influ-

ence the cell and cytokine (IL-6, IL-8, TNF) response

assessed in induced sputum. The postexposure

increase in exhaled NO levels was attenuated by

salmeterol preceded by 1 week of treatment with pla-

cebo or ibuprofen, but not fluticasone.

In our previous parallel group study [6] those who

were randomized to salmeterol treatment were slightly

less responsive to methacholine prior to exposure than

those who received placebo. It was therefore dis-

cussed whether the difference in preexposure bron-

chial responsiveness may have influenced the results.

To eliminate this possible source of error we per-

formed the present study using a cross-over design.

We have previously shown that a wash out of 1 week

is enough to normalize increased bronchial respon-

siveness induced by exposure in a pig barn [28]. We

thus regard 2–3 weeks wash out, as has been adopted

in the present study, to be sufficient to eliminate pos-

sible carry over effects from the previous exposure. In

the present study we failed to reproduce our previous

data and we found that salmeterol partially did protect

against exposure-induced enhancement of bronchial

responsiveness in healthy subjects. This finding led us

to analyse pooled bronchial provocation data from

four previous studies in which bronchial responsive-

Subjects

0

2

4

6

8

10

12

14

16

Post exposure

Pre exposure

PD

20F

EV

1

(c)

0 2 4 6 8 10 12 14 160

2

4

6

8

10

12

14

16

PD

20F

EV

1

(pre

an

d p

ost

exp

osu

re d

iffe

ren

ces)

Pre exposure PD20FEV1

y = 0.94x –0.10

r2 = 0.995

PD

20F

EV

1(p

re a

nd

po

st e

xpo

sure

do

ub

ling

s)

0 2 4 6 8 10 12 14 160

1

2

3

4

5

6

7

Pre exposure PD20FEV1

(a) (b)

y = 0.12x + 2.72r2 = 0.29

Log2 PD20FEV1 before exposure

Log 2

PD

20F

EV

1 af

ter

expo

sure

y = 0.58x –2.78

r2 = 0.55

(d)

–5

–3

–1

1

3

5

–5 –3 –1 1 3 5

Fig. 5 (a) Relation between pre-exposure PD20FEV1 and exposure-induced doubling enhancement of bronchial responsivenessin 47 healthy subjects. r = 0.54. (b) Relation between pre-exposure PD20FEV1 and the difference between pre- and postexpo-sure PD20FEV1 in 47 healthy subjects. r = 0.997. (c) PD20FEV1 before and after 3 h of exposure in a pig barn in 47 healthysubjects. Filled circles represent preexposure and open circles postexposure PD20FEV1 in the same subject along a vertical line.Pre-exposure PD20FEV1 was <0.56 mg in five subjects. Postexposure PD20FEV1 was >0.56 mg in eight subjects. (d) Relationbetween pre- and postexposure PD20FEV1 in 47 healthy subjects. Data are log2 transformed. r = 0.76.



ness was studied before and after exposure in 47

healthy subjects who were not treated or protected

during 3 h of exposure in a pig barn. In these subjects

we found a weak correlation between pre- and post-

exposure bronchial responsiveness and bronchial

responsiveness after exposure was almost identical in

all subjects irrespective of the pre-exposure level

and there was an almost perfect correlation between

pre- and postexposure difference in PD20FEV1 and

preexposure PD20FEV1. Thus, at baseline, i.e. before

exposure, bronchial responsiveness to methacholine in

these 47 healthy subjects varied within a wide range

(PD20FEV1 from 0.14 mg to >15 mg) and 42 of the

47 subjects had normal PD20FEV1 (i.e. > 0.26 mg for

women and >0.56 mg for men based on more than

200 healthy subjects). From these data it is obvious

that, following exposure to a strong pro-inflammatory

stimulus such as organic dust in a pig barn, bronchial

responsiveness to methacholine is enhanced up to a

certain level irrespective of pre-exposure bronchial

response. As approximately the same level was

reached in all subjects we conclude that the increase

in bronchial responsiveness to methacholine following

exposure in a pig barn represents what maximally can

be achieved in healthy subjects. The pre- to postexpo-

sure difference will become larger if pre-exposure

PD20FEV1 is high.

In our previous parallel group study the salmeterol-

treated group started at a higher PD20FEV1 than did

the placebo group and we found no difference of

the fall in PD20FEV1 between the two groups. From

the above discussed retrospective data it is concluded

that both the placebo group and the salmeterol group

in the previous study would have ended up at the same

postexposure level of bronchial responsiveness if no

treatment had been given. Reanalysis of these data

showed that the postexposure PD20FEV1 is somewhat

higher after salmeterol administration, both after one

single dose and after 1 week of treatment, indicating a

protective effect of salmeterol. In the present study a

cross over design was used, which eliminated the bias

posed by different pre-exposure PD20FEV1.

The protection of salmeterol in the present study was

not complete which is most likely because of the time

interval between inhalation of the drug and the meth-

acholine challenge (8 h); the effect of salmeterol had

probably weaned off at the time of the bronchial prov-

ocation. This interpretation is to some extent supported

by a previous study in which the protective effect of

salmeterol on methacholine-induced bronchoconstric-

tion was slightly diminished at 8 h compared with 2 h

after drug inhalation [6]. The uncomplete protection

offered by salmeterol could also be related to the fact

that salmeterol is a partial agonist and may therefore

not be capable of complete protection against expo-

sure-induced increase of bronchial responsiveness.

The subjects exhibited a postexposure neutrophilic air-

way inflammation and pretreatment with fluticasone

did not influence the inflammatory response in the

present study. This is in concordance with the finding

that asthma patients with predominantly neutrophilic

airway inflammation respond worse to steroid therapy

than do patients with predominantly eosinophilic air-

way inflammation [29]. Furthermore, a slight increase

of exhaled NO following exposure was found only

after placebo and fluticasone treatment in the present

study, and was thus not attenuated by the steroid. This

observation diverges from what has been described in

asthmatics with eosinophilic inflammation, where fluti-

casone treatment lowers the level of exhaled NO [30].

The effect of inhaled steroids on exhaled NO thus var-

ies depending on the type of airway inflammation.

None of the treatments in the present study influenced

the elevation of body temperature and cytokine levels

in blood and sputum observed after exposure. This is

in contrast with our earlier study where postexposure

plasma IL-6 levels and body temperature were signifi-

cantly lower after fluticasone treatment than after pla-

cebo [1]. This discrepancy is likely to be explained

by the different levels of airborne endotoxin, which

were considerably higher in the previous than in the

present study.

Based on the finding that salmeterol did not protect

against pig barn exposure-induced enhancement of

bronchial responsiveness [6], we speculated that pro-

inflammatory cytokines such as IL-1 and TNF may

induce heterologous desensitization of b2-adrenocep-



tors through formation of prostaglandin E2 [7, 8].

Therefore we administrated ibuprofen in order to find

out whether this drug was able to enhance and

thereby restore the effect of salmeterol. However, as

we were not able to confirm the lack of effect of

salmeterol this hypothesis became redundant. One

could speculate that the finding of only a partial pro-

tection offered by salmeterol may support such

a hypothesis. The lack of effect of ibuprofen does,

however, not support this speculation.

In conclusion, we found that one single dose of

salmeterol partially protects against the increased

responsiveness to methacholine following organic dust

exposure in healthy subjects but does not significantly

influence the inflammatory response to exposure in a

pig house. In addition, 1 week pretreatment with fluti-

casone or ibuprofen had no effect on the airway

responses and did not alter the effect of salmeterol.

We also conclude that exposure leads to an enhance-

ment of bronchial responsiveness up to a certain max-

imal level which is similar in all subjects and almost

totally unrelated to pre-exposure level of bronchial

responsiveness.

Conflict of interest statement

Kjell Larsson has, during the last 5 years, on one or

more occasion served in an advisoray board and ⁄orserved as speaker and ⁄or participated in education

arranged by AstraZeneca, GlaxoSmithKline, Boehrin-

ger Ingelheim and Pfizer.

Acknowledgements

Fluticasone proprionate, salmeterol and placebo were

generously provided by Glaxo Smith Kline, UK. The

study was supported by grants from The Swedish

Heart-Lung Foundation and Karolinska Institutet. The

kind support from the staff at Upppig swine confine-

ment facilities and the technical assistance of Mari-

anne Olsson, Ida von Scheele, and Britt-Marie

Sundblad is gratefully acknowledged. The authors

also would like to thank Ingrid Delin and Ewa Selg

for valuable discussions concerning PGE2.

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Correspondence: Karin Strandberg, Lung and Allergy Research,

The National Institute of Environmental Medicine, Karolinska Insti-

tutet, Box 287, SE-171 77 Stockholm, Sweden.

(fax: +46 8 300 619; e-mail: [email protected]).



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Fluticasone and ibuprofen do not add to the effect of salmeterol on organic dust-induced airway...

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