Effects of Smoking Cessation on Lung Function and Airway Inflammation in Smokers with Asthma

Effects of smoking cessation on lung function and airway inflammation

in smokers with asthma

Rekha Chaudhuri l, Eric Livingston l, Alex D McMahon 2, Jane Lafferty l,

Iona Fraser 3, Mark Spears l, Charles P McSharry 3, Neil C Thomson l

Departments of Respiratory Medicine l and Immunology 3, University of Glasgow

and Robertson Centre for Biostatistics2, Glasgow, UK

Correspondence:

Professor Neil C Thomson

Department of Respiratory Medicine

Division of Immunology, Infection and Inflammation

University of Glasgow and Western Infirmary

Glasgow, G11 6NT Scotland, UK

Telephone: 44-141-211-3241 Fax: 44-141-211-3464

E- mail: [email protected]

Funded by: Asthma UK, Scottish Council for Postgraduate Medical and Dental

Education and Chief Scientist Office, Chest Heart and Stroke, Scotland.

Running head: Smoking cessation in smokers with asthma

Descriptor number: 64

Word count: 3035

"This article has an online data supplement for the methods section, which is accessible

from this issue's table of content online at www.atsjournals.org"

AJRCCM Articles in Press. Published on April 27, 2006 as doi:10.1164/rccm.200510-1589OC

Copyright (C) 2006 by the American Thoracic Society.

1

ABSTRACT

Rationale: Active smoking in asthma is associated with worsening of symptoms, accelerated

decline in lung function and impaired response to corticosteroids.

Objectives: To examine the short-term effects of smoking cessation on lung function, airway

inflammation and corticosteroid responsiveness in smokers with asthma.

Methods and measurements: Smokers with asthma were given the option to quit or continue

smoking. Both groups underwent spirometry and induced sputum at baseline and at one, three

and six weeks. Cutaneous vasoconstrictor response to topical beclometasone, airway response to

oral prednisolone and sensitivity of peripheral blood lymphocytes to corticosteroids were

measured before smoking cessation and at six weeks.

Main results: Of 32 subjects recruited, 11 opted to continue smoking (smoking controls).

Twenty-one subjects opted for smoking cessation of whom ten quit smoking for six weeks (quit

group). Comparing quitters with controls at six weeks, the mean (Cl) difference in FEV1 was

407 ml (21,793), p=0.040; and the proportion of sputum neutrophils reduced by -29 (-51, -8),

p=0.039. Total cutaneous vasoconstrictor response score to topical beclometasone improved

following smoking cessation with a mean (CI) difference of 3.56 (0.84, 6.28), p=0.042

comparing quitters with control smokers. There was no change in airway corticosteroid

responses after smoking cessation.

Conclusions: Six weeks after smoking cessation, smokers with asthma achieved considerable

improvement in lung function and a fall in sputum neutrophil count compared to smokers who

continued to smoke. These findings highlight the importance of smoking cessation in asthma.

Word count: 236

Key words: Smoking cessation; smoking; asthma; lung function; airway inflammation

2

INTRODUCTION

Active cigarette smoking has detrimental effects on asthma morbidity. Compared to non-

smokers with asthma, smokers have more severe symptoms (1, 2), increased rates of

hospitalization (3), accelerated decline in lung function (4, 5) and impaired therapeutic responses

to inhaled (6, 7) and oral corticosteroids (8). Furthermore, the prevalence rates for cigarette

smoking in individuals with asthma are similar to that of the general population (9), and in many

developed countries over 20% of adults with asthma are active smokers (1, 2, 9-12). Particularly

high rates have been noted in adults presenting to hospital emergency departments with acute

asthma (3). There is limited information on the effects of cigarette smoking on airway

inflammation in asthma (13). Sputum neutrophil counts are reported to be increased in heavy

smokers with mild asthma compared to non-smokers with asthma (14), and sputum

concentrations of cytokines such as IL-8 are raised (14) and others such as IL-18 are suppressed

(15) in smokers with asthma.

Despite its importance, there is limited published data on the effects of smoking cessation on

symptoms, lung function and corticosteroid responsiveness in smokers with asthma. In an

uncontrolled study, seven subjects who quit smoking for a week showed improvements in

symptoms and lung function (16). In a longer term study, a prospective cohort of smokers with

asthma reported improvements in symptoms and bronchial hyperreactivity after four months of

smoking cessation (17). In ten subjects with asthma who were ex-smokers for at least a year, the

therapeutic response to oral corticosteroids was midway between that of current smokers and

never smokers; suggesting that smoking cessation may partially restore corticosteroid

responsiveness (8).

3

The effect of smoking cessation on airway inflammation in healthy smokers, shows a dose-

dependent relationship between smoking and airway inflammation (18). In these subjects, two

months smoking reduction lead to a significant fall in bronchoalveolar lavage neutrophil and

macrophage counts (19). In contrast to the improvement in airway inflammation found in

healthy smokers after smoking cessation, there is minimal change in airway inflammation in

patients with COPD after quitting smoking (20-22), and the effect of smoking cessation on

airway inflammation in smokers with asthma is not known.

Our hypothesis is that smoking cessation improves lung function, reduces airway inflammation

and restores corticosteroid responsiveness in smokers with asthma. The aim of the study was to

prospectively study smokers with asthma who successfully quit smoking and compare outcome

measures with asthmatic smokers who continue to smoke; measurements included lung

function, asthma control score, induced sputum cell counts and mediator levels. A secondary

aim was to assess the airway, cutaneous and lymphocyte sensitivity to corticosteroids after

smoking cessation. Some of the results have been reported in abstract form (23).

METHODS

Subjects

Smokers with asthma [ATS criteria, (24)] aged 18-60 years, baseline FEV1 ≤85% predicted, ≥

15% reversibility of FEV1 after nebulised albuterol, a smoking history of ≥ 10 pack years and

currently smoking ≥ 10 cigarettes/day were recruited. The study was approved by the West

Glasgow Ethics Committee.

Study design

This was a prospective, controlled study. Airway corticosteroid sensitivity to oral prednisolone,

40 mg daily for 14 days, was assessed in all subjects by change in pre-albuterol FEV1, asthma

4

control score, morning and evening PEF, daily morning and night symptoms and use of β-

agonist inhalers. Subjects were recalled to discuss smoking cessation, 2-12 months after the

corticosteroid trial. Spirometry, induced sputum, exhaled nitric oxide (FENO), exhaled carbon

monoxide (CO), asthma control score, skin vasoconstrictor response to beclometasone, serum

cotinine and peripheral blood lymphocyte proliferation assay (LP) were performed. All subjects

were given the option to quit or continue smoking. Both groups were issued diary cards. Visits

were arranged one, three and six weeks later. Following the six-week visit, both groups

received a second course of oral prednisolone 40mg daily for 14 days, with similar end-points

used to compare results. Spirometry, exhaled gases and asthma control score were recorded at

all visits; induced sputum at three and six weeks; cutaneous vasoconstrictor test and LP at six

weeks. Patient adherence to smoking cessation was accepted by patient history.

Measurements

ATS asthma impairment score (25) and a validated asthma control questionnaire (26) were

recorded. Patients maintained a validated diary card (27), recording morning and night PEF

(Mini-Wright, Clement Clarke, Harlow, UK), daytime symptoms, night awakenings, use of

inhaled rescue medication and study tablet consumption. Spirometry was measured with a dry

spirometer (Vitalograph Ltd, Buckingham, UK). Sputum was induced as previously described

(14, 28, 29). FENO and CO were measured using a chemiluminescence analyzer (Logan

Research Ltd, Rochester, UK) (30) and serum cotinine by enzyme immunoassay (Cozart

Bioscience Ltd, Abingdon, UK). Treatment compliance was assessed by tablet count.

Cutaneous vasoconstrictor response to topical beclometasone was measured as described

previously (31, 32). Concentrations of 0, 1, 3, 10, 30, 100, 300 and 1000 µg/ml were applied to

the skin in a random double-blind manner and the degree of blanching assessed visually after 18

hours by a single trained observer. Blanching at each concentration was graded according to a 4-

5

point scale [0-3] and a total score calculated [0-24]. Sensitivity of peripheral blood T-

lymphocytes to glucocorticoids was assessed in a functional assay as described previously (33).

The percentage suppression at the final concentration of corticosteroid used was defined as the

maximum inhibitory dose [Imax %].

Statistical analysis

Baseline characteristics were compared by chi-squared, Wilcoxon and t-tests. Response to

smoking cessation on lung function, diary data, induced sputum, mediator levels, skin

vasoconstrictor response and oral prednisolone trials for smokers versus never smokers with

asthma was assessed by Analysis of Covariance models that adjusted each factor by its baseline

measure. Correlations were assessed by Spearman’s rank correlations. Only those subjects who

completed the study up to the six week visit were included in the analysis. Significance at a level

of 5% was considered for the primary end-point; the change in FEV1 at six weeks. Tests were

performed using SAS version 9.0 (Cary, NC, USA).

RESULTS

Recruitment

The flow of participants through the study is depicted in Figure 1. 11/32 subjects opted to

continue smoking (smoking controls). Of the 21 who opted to attempt quitting smoking, 14

achieved 1 week off and 10 completed six weeks of smoking cessation.

Baseline characteristics

There were no differences in age, duration of asthma, equivalent dose of inhaled beclometasone,

asthma control score, ATS impairment score, IgE levels, spirometry, skin vasoconstrictor test

6

score, inhibitory dose of dexamethasone maximally suppressing lymphocyte proliferation in

vitro, induced sputum cell counts and inflammatory mediators between the smoking control and

quit group. Mean (SD) baseline FEV1 percentage predicted was 68 (11) in the quit group and 64

(94) in the controls [Tables 1 and 2]. Baseline reversibility of FEV1 to inhaled albuterol [mean%

(SD)] was 25 (11) in the quit group and 19 (6) in the control group. Age, gender distribution,

atopic status, smoking history, duration of asthma and serum cotinine levels were similar in the

successful quitters and those subjects who opted for the quit group but were unable to achieve a

week off smoking (n=7). In the 10 subjects who successfully quit smoking and completed the

study, five used nicotine patches, one subject used acupuncture and four quit without any aid.

Change in lung function, asthma control and airway inflammatory markers with smoking

cessation

Lung function: The mean (SD) change in FEV1, ml in the quit group was 356 (278) at 1 week,

390 (311) at 3 weeks, and 450 (471) at 6 weeks of smoking cessation, p values 0.015, 0.009 and

0.031 respectively. There was no difference in the equivalent FEV1 measures in the smoking

control group. Comparing quitters with control smokers at six weeks’ cessation, there was a

mean improvement of 407 ml in FEV1, 15.2% in FEV1% predicted and 93L/min in PEF (Figure

2, Table 3). There was no correlation with the improvement in FEV1 and age, gender, atopy,

duration of smoking, pack years smoked, dose of inhaled corticosteroids and sputum cell counts.

Asthma control: The mean (CI) asthma control score showed an improvement at six weeks after

smoking cessation of -1.2 (-2.2,-0.2), p=0.021 (Table 3).

Induced sputum: Quitters showed a decrease in proportion of sputum neutrophils compared to

controls at six weeks [mean % difference (CI) -29 (-51, -8), p=0.013; with no change in

inflammatory mediator levels (Table 4). The mean (CI) difference for absolute neutrophil

count in sputum at three weeks was -52 (-141, 36), p=0.224 and at six weeks cessation it was

-121 (-215, -26), p=0.017. The degree of improvement in FEV1 was not related to the

7

baseline neutrophil level [r= 0.24, p=0.248] or the change in neutrophil proportion [r= -0.11,

p=0.819]. There was no difference in mediator response in those quitters who used nicotine

replacement therapy compared to those who did not.

Exhaled gases: Compared to the control smokers the quit group showed a reduction in exhaled

CO at one, three and six weeks. (Table 3). There was no change in exhaled NO levels.

Response to corticosteroids

Airway response: There was no difference in the FEV1 response to oral prednisolone while

smoking or at eight weeks after smoking cessation in the control smokers with asthma compared

to quitters. The mean (CI) change in FEV1, ml with the first prednisolone course when subjects

were smoking was 105 (-231,441), p=0.518 for the quit group versus smoking control group and

after eight weeks of smoking cessation, the difference was 190 (-121, 501), p=0.214.

Compliance with medication was 89% in controls and 100% in the quit group for the first

corticosteroid course and 89% in controls and 100% in quitters with the second course.

Cutaneous vasoconstrictor response to topical beclometasone: The mean (CI) total cutaneous

vasoconstrictor response score to topical beclometasone improved following smoking cessation

with a mean difference of 3.56 (0.84, 6.28), p=0.014; comparing quitters with control smokers.

Lymphocyte proliferation response: There was no difference in the lymphocyte proliferation

response after six weeks of smoking cessation. The mean difference (CI) Imax % for

dexamethasone suppression of lymphocyte proliferation was -9.3 (-28.4, 9.9), p=0.322 for

quitters compared to smokers and for prednisolone suppression was -4.0 (-17.1, 9.0); p=0.522.

DISCUSSION

Active cigarette smoking is known to worsen the severity of asthma (13). In this study we have

demonstrated that lung function of smokers with asthma improves by a considerable degree

8

within a week of stopping smoking. This improvement in FEV1 [mean (CI)] at one week was of

the order of 396 (129,664) ml compared to those that continued to smoke and this improvement

increased up to six weeks after smoking cessation. In addition, there was a fall in the sputum

neutrophil counts, evident at six weeks after smoking cessation.

Subjects in the quit group progressed through the study unless they admitted to smoking. This

method of assessment of smoking status is known to overestimate the number of subjects who

effectively quit smoking. Due to the short half-life, only daily measurements of carbon

monoxide or cotinine could have provided objective evidence for smoking cessation in between

clinic visits. Cotinine can be affected by nicotine replacement therapy used for cessation and

daily measurements for eight weeks were not feasible. However the reduction in exhaled carbon

monoxide in the quit group compared to controls at all visits provided objective evidence for

effective smoking cessation, at least for the previous 24-48 hours prior to each clinic

assessment. If some of the subjects in the quit group had not stopped smoking completely, the

results obtained may underestimate the extent of the improvement in lung function and

neutrophil count possible with smoking cessation.

The improvement in lung function seen following smoking cessation was clinically significant.

This demonstrates that there is a reversible component to the harmful effects of smoking on the

airways in asthma. The degree of improvement noted by smoking cessation far exceeds that of

high dose anti-inflammatory treatment, such as oral prednisolone 40 mg daily for 2 weeks,

which had no effect on lung function in smokers in our current study and in our previous work

(8). The improvement in lung function could be due to removal of the acute bronchoconstrictor

effects of cigarette smoke (34) or a reduction in the pro-inflammatory effects of cigarette smoke

on the airways (14). It is also possible that stopping smoking leads to a reduction in

corticosteroid insensitivity, as ex-smokers who had quit smoking for at least one year have a

9

better response to oral prednisolone compared to current smokers (8). Reduced histone

deacetylase activity is one of the possible mechanisms of corticosteroid insensitivity in smokers

(34), but there are no reports of measurement of HDAC levels following smoking cessation.

In asthma, there have been two previous studies looking at the effect of smoking cessation on

lung function. The first was a small uncontrolled study where 7 subjects quit smoking for a week

and showed improvements in PEF, symptoms and specific airways conductance (16). There was

a reduction in histamine airway responsiveness after 24 hours of smoking cessation (16). A

prospective cohort study compared the effects of smoking cessation, smoking reduction and

continuing smoking on asthma control and biomarkers of exposure to cigarette smoke (17). This

study showed a significant improvement in symptoms and bronchial hyperreactivity after four

months of smoking cessation, but no change in FEV1. The baseline FEV1 % predicted was much

higher than in our subjects and hence it is possible that scope for further improvement was

limited.

In our study, although the asthma control score improved, we did not obtain improvement in

symptoms in the diary card up to two months after cessation. In a study by Hillerdahl and

Rylander, two thirds of subjects with asthma who quit smoking reported no improvement or

worsening of symptoms after smoking cessation (35). A cross-sectional study of 3197

smokers, ex-smokers (stopped smoking for at least one year) and non-smokers with asthma,

reported that ex-smokers had significantly less cough, wheeze, night symptoms and sputum

production compared to current smokers, but a similar degree of shortness of breath (36). This

implies that many, but not all asthma symptoms can return to the level of never-smokers with

smoking cessation.

10

The raised sputum neutrophil count found in high intensity smokers with asthma (14, 37)

may be partly responsible for their reduced responsiveness to corticosteroids. Unlike

eosinophils, which are exquisitely sensitive to corticosteroids, neutrophils are poorly

responsive to corticosteroid therapy (37) and their survival and proliferation is promoted by

glucocorticoids (38). We demonstrated a reduction in induced sputum neutrophils with

smoking cessation, but no change in mediator levels. We are unaware of any previous

published studies that have measured airway inflammation in smokers with asthma after

smoking cessation. In healthy heavy smokers bronchoalveolar lavage neutrophil counts were

reduced two months after smoking reduction (19). In contrast, the effect of smoking cessation

on airway inflammation in COPD has shown that most inflammatory cells, including

neutrophils, persist in ex-smokers (20, 22, 39) and can even increase (40). The mechanisms

by which the neutrophil count is reduced following smoking cessation are unclear at present.

It seems unrelated to the continued presence of neutrophil chemo-attractants LTB4 and IL-8

in the airways, and may therefore be related to reduced diapedesis following the down-

regulatory effects of smoking cessation on adhesion molecules (41), perhaps in addition to

more efficient apoptosis of neutrophils by alveolar macrophages after smoking cessation (42,

43).

The short-term improvement in FEV1 was not related to the change in neutrophil count, which

might suggest that the removal of bronchoconstrictor effects of cigarette smoke is more

important than neutrophils themselves; but the small number of subjects studied may explain our

findings. The levels of the inflammatory mediators in induced sputum did not change at six

weeks following smoking cessation. This result is similar to the finding in COPD, where IL-8

levels in sputum were similar in smokers and ex-smokers (44) and chronic bronchitis, where

BAL levels of TNF-alpha were unaltered by smoking cessation (22). It is possible that clinical

improvement may precede changes in inflammation, as in the third national health and nutrition

11

examination survey (NHANES III), inflammatory markers (CRP, white cell count, fibrinogen

and albumin) resolved more slowly than traditional cardiovascular risk factors (45).

Smoking cessation did not improve corticosteroid responsiveness measured by changes in FEV1

and PEF. However, there was a large improvement in baseline lung function with smoking

cessation and it is possible that this reduced the scope for further improvement in lung function,

after corticosteroids. In a previous study, ex-smokers with asthma who had quit smoking for at

least a year showed an improvement in morning PEF, but not FEV1 after high dose oral

prednisolone compared to placebo (8).

The skin vasoconstrictor response to topical beclometasone (32, 46) is based on the ability of

corticosteroids to cause transient vasoconstriction and skin blanching, has been used as a

screening test to determine the relative anti-inflammatory potency of inhaled corticosteroids (47)

and as an index of systemic sensitivity to corticosteroids (31). Objective methods of detecting

glucocorticoid-induced skin blanching have been compared to the visual scoring system, but the

human eye has been found to be the most sensitive tool to measure dermal blanching (32). The

cutaneous vasoconstrictor test for corticosteroid sensitivity showed an improvement after six

weeks of cessation implying some restoration in peripheral corticosteroid sensitivity within this

period. The mechanism for skin vasoconstriction with corticosteroids is not known, but a

possible explanation is that glucocorticoids increase the sensitivity of vascular smooth muscle to

the vasoconstrictor effects of noradrenaline (48). The blanching is possibly mediated through

glucocorticoid receptors, since oral administration of RU 486, a glucocorticoid receptor

antagonist, can abolish this response (49). Smoking causes vascular dysfunction but the additive

effect of nicotine on corticosteroids has not been studied in skin vasculature. The lymphocyte

proliferative response did not alter with smoking cessation in our study. A lack of correlation

between different tests of tissue sensitivity to corticosteroids has been reported previously in

12

health human volunteers (50).

In conclusion, in smokers with asthma, improvement in lung function occurs as early as one

week after smoking cessation with a further improvement up to six weeks. There is a reduction

of sputum neutrophil percentage after six weeks of smoking cessation but no change in common

inflammatory mediator levels. These findings highlight the importance of smoking cessation in

asthma.

13

ACKNOWLEDGEMENTS

We are grateful to Kathy McFall, medical illustration department, for assistance with the

figures.

14

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Norepinephrine Uptake by Human Bronchial Arterial and Rabbit Aortic Smooth Muscle

Cells. Am J Respir Cell Mol Biol 2001;25(4):500-506.

49. Gaillard R, Poffet D, Riondel A, Saurat J. RU 486 inhibits peripheral effects of

glucocorticoids in humans. J Clin Endocrinol Metab 1985;61:1009-1011.

50. Hearing S, Norman M, Smyth C, Foy C, Dayan C. Wide variation in lymphocyte steroid

sensitivity among healthy human volunteers. J Clin Endocrinol Metab 1999;84(11):4149-

19

4154.

51. Juniper E, Svensson K, Mork A, Stahl E. Measurement properties and interpretation of

three shortened versions of the asthma control questionnaire. Resp Med 2005;99:553-558.

52. Johansson S, Hourihane JB, Bousquet J, et al. A revised nomenclature for allergy: An

EAACI position statement from the EACCI nomenclature task force. Allergy

2001;56:813-849.

20

Figure Legends

Figure 1

Flow of subjects through the study

Figure 2

Mean (95% CI) difference between quitters and control asthmatic smokers in change in FEV1 (ml)

compared to baseline.

21

Table 1: Demography at baseline mean (SD) in smokers with asthma who continued smoking

compared to smokers with asthma who quit smoking.

Asthmatic smokers

Test Smoking controls Quit smoking group

(n=10) (n=10)

Age, years 45.7 (6.8) 49.5 (9.3)

Gender, M/F 3/7 5/5

Asthma duration, yrs 21.6 (16.8) 12.0 (7.2)

Smoking history

pack years 36.5 (11.7) 36.1 (21.2)

cigarettes/day 25.5 (4.4) 22.7 (8.9)

years smoked 28.3 (6.4) 30.1 (7.9)

Inhaled corticosteroids, n (%) 8 (80%) 6 (60%)

Equivalent beclometasone

dose (mcg) median(IQR) 500(400-4000) 1800(1000-2000)

Total IgE, IU/ml, median (IQR) 161 (86-239) 56 (20-135)

Specific IgE +ve, % 75% 38%

Serum cotinine, ng/ml 237 (126) 290 (232)

FEV1, L pre-albuterol 1.90 (0.70) 2.15 (0.53)

FEV1 % predicted, pre albuterol 68 (11) 64 (9)

FEV1, L post-albuterol 2.12 (0.85) 2.27 (0.57)

FVC, L pre-albuterol 3.26 (1.05) 3.20 (1.08)

FEV1/FVC pre-albuterol 59 (10) 63 (9)

FEV1/FVC post-albuterol 63 (12) 66 (10)

FEF 25-75 pre-albuterol, % predicted 26 (13) 28 (10)

PEF during spirometry, L/min 300 (114) 327 (98)

Asthma control score 2 (1) 3 (1)

ATS impairment score 4.7 (1.7) 5.1 (1.7)

22

Morning PEF from diary, L/min 334 (129) 370 (99)

Evening PEF, from diary, L/min 376 (99) 377 (81)

Rescue inhaler use, puffs 3 (3) 4 (3)

Daytime symptom score 8 (5) 9 (3)

Night symptom score 1 (1) 1 (1)

This table compares asthmatic smokers who continued to smoke with those who successfully quit smoking for 6 weeks. P > 0.05 for all baseline data.

23

Table 2: Baseline induced sputum cell counts, mediators in sputum exhaled gases and tests of

corticosteroid responsiveness.

Smokers with asthma

Test Smoking controls Quit smoking group

(n=10) (n=10)

Induced sputum cell counts [median (IQR)].

Total Cell count x106 3 (2-4) 3 (2-4)

Neutrophils % 49 (27-54) 41 (27-68)

Eosinophils % 2 (1-3) 1 (1-2)

Macrophages % 44 (34-54) 45 (25-65)

Lymphocytes % 2 (1-3) 1 (1-3)

Bronchial epithelial cells% 4 (2-5) 5 (1-6)

Viability 84 (81-86) 78 (72-88)

Sputum supernatant mediators [median (IQR)].

IL-8, ng/ml 26 (25-26) 26 (25-26)

MPO, ng/ml 124 (40-215) 166 (63-200)

LTB4, pg/ml 2783 (635-3875) 757 (445-1862)

ECP, ng/ml 177 (60-562) 102 (54-144)

Exhaled gases [mean (SD)]

Exhaled CO, ppm 23.5 (10.4) 17.6 (6.6)

Exhaled NO, ppb 3.9 (1.9) 7.6 (4.9)

Tests of corticosteroid responsiveness [mean (SD)]

Skin vasoconstrictor test score 6.3 (2.5) 4.8 (3.4)

LP dexamethasone, Imax % 45 (31) 50 (43)

LP prednisolone, Imax % 12 (31) 19 (55)

P > 0.05 for all baseline data.

Table 3: Quit smoking group versus smoking controls with asthma mean difference (CI) compared to baseline

Test Mean difference (CI) and p value 1 week cessation 3 weeks cessation 6 weeks cessation 8 weeks cessation, post steroids

∆ FEV1 pre albuterol, ml 396 (129,664)† 397 (67,727)* 407 (21,793)* 656 (228,1084) †

∆ FEV1 % predicted 13.9 (3.8,24.0)* 16.2 (3.4,29.0)* 15.2 (4.0,26.4)* 22.1 (8.2,36.0) †

∆ FVC, ml pre-albuterol 765 (200,1330)* 713 (92,1335)* 856 (274,1437) † 838 (294,1381) †

∆ FEF 25-75 pre-albuterol, ml 128 (-307,562) 261 (-120,642) 36 (-342,414) 477 (-80,1034)

∆ PEF during spirometry, 71 (-0.8,143)* 66 (-2,133)* 93 (33,153) † 98 (22,175)*

L/min, pre-albuterol

∆ Asthma control score -0.1 (-1.0,0.7) -0.6(-1.5,0.3) -1.2 (-2.2,-0.2)* -0.4 (-1.4,0.6)

∆ Diary morning PEF, L/min 12(-80,103) 52 (-5,110) 41 (-12,904) 50 (-10,109)

25

∆ Evening PEF, L/min 10 (-85,106) 43 (-9,94) 49 (-2,100) 56 (-5,116)

∆ Daytime symptom score -1 (-5,4) -3 (-8,2) -2 (-7,2) -1 (-7,5)

∆ Night symptom score 0 (-1,1) 0 (0,1) -1 (-1,0)* 0 (-1,1)

∆ Reliever inhaler use, puffs 1 (-2,4) -1 (-4,2) -1 (-4,1) -1 (-5,3)

∆ Exhaled nitric oxide, ppb -2.0 (-5.7,1.7) 6.4 (-1.3,14.1) -1.2 (-4.9,2.6) 0.4 (-1.6,2.4)

∆ Exhaled CO, ppm -15.8 (-25,-6.1) † -13.1 (-22.7,-3.5) † -15.0 (-23.7,-6.2) † -15.6 (-24.9,-6.3) †

All analyses done by ANCOVA.. *=p ≤ 0.05, † = p ≤ 0.01 For lung function data, n=10 for controls and quitters. For diary data, n=5-8 for controls and 5-9 for quitters.

26

Table 4: Change mean (CI) in induced sputum cell counts and mediators in smokers with asthma following smoking cessation compared to

smoking controls at 3 and 6 weeks after smoking cessation.

3 weeks cessation 6 weeks cessation

Induced sputum cell counts

∆ Total cell count x106 -1.31 (-5.0,2.4) -4.2 (-9.7,1.2)

∆ Neutrophils % -8.1 (-30.1,13.9) -29.1 (-50.8,-7.5) †

∆ Eosinophils % 1.4 (0,2.7) 0.1 (-1.8,1.9)

∆ Macrophages % 6.2 (-15.7,28.0) 21.6 (4.2,39.1)*

∆ Lymphocytes % 0.5 (-0.6,1.4) 1.1 (-1.0,3.1) ∆ Bronchial epithelial cells % 1.0 (-1.6,3.6) 7.2 (-4.0,18.4) ∆ Sputum supernatant mediators

∆ IL-8, ng/ml -2.1 (-6.2,2.0) 0.4 (-0.8,1.6)

∆ MPO, ng/ml -69 (-220,82) -24 (-214,167)

∆ LTB4, pg/ml 453 (-1154,2060) -953 (-2935,1028)

∆ ECP, ng/ml 50 (-126,227) -19 (-199,160)

Analyses performed using ANCOVA. *=p ≤ 0.05, † = p ≤ 0.01 At 3 weeks, n=9 for cell counts and 8-9 for the different mediators. At 6 weeks, n=8 for controls and 10 for quitters for cell counts and 7 controls and 6-10 for the different mediators

Figure 1

32 smokers with asthma recruited

First oral prednisolone trial n=25. Seven subjects refused/ had medical conditions that precluded high dose oral steroids.

21 opted for smoking cessation

1 week follow up visit: 11 subjects attended

11 opted to be continue smoking (control smokers)

3 week follow up visit: 12 achieved 3 weeks cessation,

2 withdrawn as they had re-started smoking

6 week follow up visit: 10 subjects attended

2 withdrawn as they had re-started smoking

1 week follow up visit: 14 succeeded in smoking cessation

7 withdrawn as unable to quit

Second oral prednisolone trial n=19 (10 in the quit group and 9 controls). 1 subject refused high dose oral steroids.

3 week follow up visit: 10 subjects attended,

1 drop-out due to leg ulcers

6 week follow up visit: 10 subjects attended

28

Figure 2

29

Effects of smoking cessation on lung function and airway inflammation in

smokers with asthma

Rekha Chaudhuri l, Eric Livingston l, Alex D McMahon 2, Jane Lafferty l,

Iona Fraser 3, Mark Spears l, Charles P McSharry 3, Neil C Thomson l

Online Data Supplement

30

METHODS

Subjects

Smokers with asthma aged 18-60 years were recruited. The study was approved by the West

Glasgow Ethics Committee and all participants gave written informed consent. Asthma was

diagnosed by ATS criteria (E1). Subjects had a baseline FEV1 ≤85% predicted and

demonstrated ≥ 15% reversibility of FEV1 after nebulised albuterol. Smokers with ≥ 10 pack

years smoking history and currently smoking ≥ 10 cigarettes/day were included.

Study design

This was a prospective, controlled study performed in two parts. Initially, smokers with asthma

received 40 mg of oral prednisolone for 14 days after performing baseline spirometry and

reversibility to nebulized albuterol. End-points used to assess airway corticosteroid sensitivity

were pre-albuterol FEV1, asthma control score, change in morning and evening PEF, daily

morning and night symptoms and reduction in the use of β-agonist inhalers. Subjects were then

recalled to discuss smoking cessation, two to twelve months after the corticosteroid trial.

Spirometry, induced sputum, exhaled nitric oxide, exhaled carbon monoxide, asthma control

score, cutaneous vasoconstrictor test were performed and peripheral blood taken for lymphocyte

proliferation functional assay. Serum cotinine was measured to confirm smoking status. All

subjects were given the option to quit smoking or continue smoking. The quitting and continued

smoking groups were issued diary cards to record morning and night-time peak flows,

symptoms and use of reliever inhaler. The smoking cessation group were allowed to choose any

method of support that would help them to quit (e.g. nicotine replacement, zyban or self-control

alone). Visits were arranged one, three and six weeks later. After the six weeks cessation visit,

both groups received oral prednisolone 40mg daily for 14 days, as in the initial phase of the

study, with similar end-points used to compare results. Spirometry, exhaled gases and asthma

31

control score were recorded at all visits. Induced sputum was performed at three and six weeks;

cutaneous vasoconstrictor test and blood lymphocyte proliferation were performed at six weeks.

Exhaled carbon monoxide was measured at all visits. Quitting smoking was accepted by self-

report from the patient. If a subject in the quit group had a lapse that lasted a day, they were

allowed to continue in the study, but their next appointment was postponed by a week. If the

smoking lapse lasted for two or more days, they were encouraged not to smoke, but were

withdrawn from the study.

Measurements

Asthma severity was scored according to the ATS asthma impairment score (E2) and asthma

control was measured using a validated questionnaire with a range from 0 to 6 for increasing

severity and a reduction of 0.5 as a minimal important difference (E3,E4). Patients maintained a

validated diary card (E5), recording morning and night PEF (Mini-Wright peak flow meter,

Clement Clarke, Harlow, UK), daytime symptoms (range 0-6, for increasing severity) and night

awakenings (range 0-3, for increasing severity), use of inhaled rescue medication and study

tablet consumption. Total serum IgE and specific IgE to house dust mite, grass pollen and cat

dander were measured by enzyme linked immunoassay (Unicap, Pharmacia Ltd, Milton Keynes,

UK) (E6). Spirometry was measured with a dry spirometer (Vitalograph Ltd, Buckingham, UK).

Sputum induction with 3% hypertonic saline was performed using a modification of the method

described by Pin et al (E7,E8,E9). Eosinophil cationic protein (ECP), interleukin-8 (IL-8),

myeloperoxidase (MPO) and leukotriene B4 (LTB4) were measured in the fluid-phase of induced

sputum samples. ECP was measured using a sandwich ELISA assay (Caltag Medsystems,

Buckinghamshire, UK) with a sensitivity of 0.125ng/ml, and an inter-assay variability <10%, an

intra-assay variability of <5%, and a recovery in spiked samples of 105+/-9%. LTB4 was

measured using a sandwich ELISA assay (R&D Systems, Oxon, UK) with a sensitivity of

5.63pg/ml, an intra-assay variability of 9.3%, and a recovery in spiked samples of 114.1%.

32

Cross-reactivity with other compounds was:- 6-trans-12-epi-LTB4 (5.5%), 6-trans-LTB4 (4.9%),

12-epi-LTB4 (0.94%), and < 0.2% for the following PGE2, PGF2, 20-OH-LTB4, 20-COOH-

LTB4, LTC4, LTD4, LTE4, 5(S)-HETE, 12(S)-HETE and 15(S)-HETE. MPO was measured by

ELISA (Metachem Diagnostics, Northampton, UK) with a sensitivity of 0.13ng/ml, an intra-

assay variability of <5.4%, an inter-assay variability of <8.8%, and a recovery in spiked sputum

samples of 94%. Cross reactivity with related human compounds was:- erythropoietin 1.1%,

elastase 0.2%, lactoferrin 0.2%, thyroid peroxidase < 0.1%, eosinophil-derived neurotoxin <

0.1%. IL-8 was measured by fluorescent-linked immunoassay (Biosource, Nivelles, Belgium) as

part of a multiplex kit (Human inflammatory 5-plex), with a sensitivity of 8 pg/ml, an intra-assay

variability of <5%, an inter-assay variability of <10%.

Exhaled nitric oxide and carbon monoxide were measured using a chemiluminescence analyzer

(Logan Research Ltd, Rochester, UK) (E10). Subjects who were smoking were asked to abstain

for an hour prior to their visit. Serum cotinine was measured using an enzyme immunoassay

(Cozart Bioscience Ltd, Abingdon, UK) to confirm the smoking status of all subjects.

Compliance was assessed by counting the numbers of tablets remaining after each treatment

period.

The cutaneous vasoconstrictor response to topical beclometasone was measured as described

previously (E11,E12), with minor modifications. Beclometasone dipropionate (Sigma, Poole,

UK) was dissolved in 95% ethanol to concentrations of 1, 3, 10, 30, 100, 300 and 1000 µg/ml. A

control solution of 95% ethanol was used. Solutions were stored at 4°C and used within 2

weeks of preparation. Test sites were outlined by the application of adhesive tape in which

holes, 2cm in diameter, had been cut. Solutions were randomly allocated a letter (A-H) by staff

not involved in the application of solutions or the reading of the test, and solutions applied in

order, A-H. The test was not unblinded until the completion of the study. After evaporation of

33

the diluent, the sites were occluded (for 16-18 hours) with plastic film to enhance percutaneous

absorption of the beclometasone. A tubular bandage (Tubigrip; Seton Healthcare Group plc,

Manchester, UK) was applied to the forearm to attenuate any changes in ambient temperature.

The following morning the adhesive tape and plastic film were removed, and the degree of

blanching assessed 1-2 hours later. The test sites were examined in standard lighting conditions

and given a blanching score by a single trained observer who did not know the order of the

applications of the solutions. Blanching at each concentration was graded according to a 4-point

scale: 0 = no blanching; 1 = faint blanching; 2 = obvious blanching not extending outwith the

test site; 3 = intense blanching extending over the margin of the test site. Addition of individual

concentration scores gave a total score [range 0-24]. A high score indicated a high degree of

corticosteroid sensitivity.

The sensitivity of peripheral blood T-lymphocytes to glucocorticoids was assessed in a

functional assay as described previously (E13) at the same two visits as the skin test. The

percentage suppression at the final concentration of corticosteroid used (dexamethasone or

prednisolone), termed Imax % in this study was compared between the two groups.

Statistical analysis

Baseline characteristics were compared by chi-squared tests, Wilcoxon tests and t-tests.

Diary data was analysed using the last three days of each period. The first three days of diary

card data were used as baseline values. The response to smoking cessation on lung function,

diary data, induced sputum, mediator levels, skin vasoconstrictor response and oral

prednisolone trials for smokers versus never smokers with asthma was assessed by Analysis

of Covariance models that adjusted each factor by its baseline measure. Correlations were

assessed by Spearman’s rank correlations. Only those subjects who completed the study up

to the six week visit were included in the analysis. Significance at a level of 5% was

34

considered for the primary end-point; the change in FEV1 at six weeks. A sample size of 10

in each group will have 80% power to detect a difference in means of 300.0 ml assuming

that the common standard deviation is 224.2 using a two group t-test with a 0.050 two-sided

significance level. Tests were performed using SAS version 9.0 (Cary, NC, USA).

35

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Additional Results: Table E1: FEV1 [mean (SD)] while smoking (first steroid trial) and after cessation in quitters and controls (second steroid trial). ` First steroid trial Second steroid trial P value for (all subjects smoking) difference of n=10 in each group (quitters n=10, controls n=9) differences comparing both steroid trials Pre-steroid Post steroid p value Pre-steroid Post steroid p value FEV1 pre-albuterol, L Control group 1.9 (0.8) 2.0 (0.7) 0.380 2.0 (0.8) 1.9 (0.7) 0.095 0.194 Quit group 1.9 (0.4) 2.1 (0.6) 0.338 2.4 (0.7) 2.4 (0.8) 0.585 0.620 FEV1% predicted Control group 65 (12) 68 (10) 0.210 66 (12) 64 (14) 0.280 0.253 Quit group 65 (15) 68 (14) 0.557 77 (14) 78 (17) 0.767 0.719