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Original Research: COPD |

Young “Healthy” Smokers Have Functional and Inflammatory Changes in the Nasal and the Lower AirwaysAirway Dysfunction in Young Smokers FREE TO VIEW

Marina Lazzari Nicola, BSc; Heráclito Barbosa de Carvalho, MD, PhD; Carolina Tieko Yoshida, BSc; Fabyana Maria dos Anjos, PhD; Mayumi Nakao, MD; Ubiratan de Paula Santos, MD, PhD; Karina Helena Morais Cardozo, PhD; Valdemir Melechco Carvalho, MD, PhD; Ernani Pinto, PhD; Sandra Helena Poliselli Farsky, PhD; Paulo Hilario Nascimento Saldiva, MD, PhD; Bruce K. Rubin, MD, MEngr, MBA; Naomi Kondo Nakagawa, PhD
Author and Funding Information

From the Department of Pathology (Mss Nicola and Yoshida and Drs Nakao, Saldiva, and Nakagawa), and Department of Physiotherapy (Mss Nicola and Yoshida and Dr Nakagawa), Communication Science and Disorders, Occupational Therapy, LIM 34, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; Department of Preventive Medicine (Dr H. B. Carvalho), and Department of Clinical and Toxicological Analysis (Drs Anjos, Pinto, and Farsky), Faculty of Pharmaceutical Sciences, Universidade de São Paulo, São Paulo, Brazil; Pulmonary Division (Dr Santos), Heart Institute, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; Fleury Medicine and Health Institute (Drs Cardozo and V. M. Carvalho), São Paulo, Brazil; and the Department of Pediatrics (Dr Rubin), Virginia Commonwealth University School of Medicine, Richmond, VA.

Correspondence to: Naomi Kondo Nakagawa, PhD, Faculdade de Medicina da Universidade de São Paulo, 455 Av. Dr. Arnaldo, room 1150, Cerqueira Cesar, São Paulo, Brazil, CEP 01246-903; e-mail: naomi.kondo@usp.br


Funding/Support: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP 13/13598-1 and 13/11401-6].

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2014;145(5):998-1005. doi:10.1378/chest.13-1355
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Background:  Smoking is responsible for most COPD. Although people with COPD often have concomitant nasal disease, there are few studies that report physiologic or inflammatory changes in the upper airways in young asymptomatic smokers. We investigated physiologic and inflammatory changes in the nasal and lower airways of young smokers and if these changes were related to smoking history.

Methods:  Seventy-two subjects aged between 18 and 35 years (32 healthy nonsmokers and 40 young smokers) participated in this study. We measured nasal mucociliary clearance (MCC), nasal mucus surface contact angle, cell counts, myeloperoxidase and cytokine concentrations in nasal lavage fluid, exhaled breath condensate (EBC) pH, and lung function.

Results:  Smokers had faster MCC, an increased number of cells (macrophages, ciliated cells, and goblet cells), increased lavage myeloperoxidase concentration, and decreased EBC pH compared with nonsmokers. There was a significant inverse relationship between pack-year smoking history and EBC pH. There were no differences in lung function or mucus surface properties comparing smokers to nonsmokers.

Conclusions:  Young adult smokers have functional and inflammatory changes in the nasal and lower airways and these correlate with smoking history. However, in these young smokers, smoking history was not associated with pulmonary function decline, probably because it is unlikely that spirometry detects early physiologic changes in the airways.

Trial registry:  ClinicalTrials.gov; No.: NCT01877291; URL: www.clinicaltrials.gov

Figures in this Article

The upper airway epithelium is the first defense of the respiratory system against inhaled toxic agents and microorganisms. This protective barrier depends on the integrity of the epithelial surface. Long-term tobacco smoking alters this barrier by oxidative stress and airway inflammation.1,2 These increase epithelium permeability,3 impair ion transport,4 deplete cilia,5,6 induce metaplasia of goblet and squamous cells, and increase mucus secretion.7 Thus, smoking may impair mucociliary clearance (MCC),8,9 leading to airway obstruction,2 decreased lung function,10 increased susceptibility to respiratory infections,11,12 and progression of COPD, particularly in predisposed long-term smokers.13 Approximately 25% of long-term smokers develop COPD, and initial lung function alterations can be observed in some smokers as young as 35 years of age14,15 with a smoking history of ≥ 20 pack-years.15 Studies in smoking adults < 35 years of age suggest that tobacco smoking is an important risk factor for chronic cough and that this is related to pack-year smoking history.16,17

On the other hand, studies of younger smokers or those with less pack-year history also suggest that “light” smoking and environmental tobacco smoke exposure is associated with faster nasal ciliary beat frequency and mucus clearance18 and that the mucociliary transportability of lower airway mucus is faster than mucus from healthy nonsmokers. This declines with longer smoking history, however, is impaired in subjects with diagnosed COPD.7 Because of the close association of COPD with nasal and sinus inflammation,19 we investigated if smokers younger than age 35 years with short pack-year smoking history have physiologic or inflammatory changes in the upper and lower airways and if these changes are related to smoking history.

Over a period of six consecutive months, we recruited subjects aged between 18 and 35 years from the Faculdade de Medicina da Universidade de São Paulo. Subjects were invited by telephone to participate in the study; the study objectives and procedures were discussed, and subjects were included in the study after obtaining written informed consent. Exclusion criteria were the inability to understand and follow commands, previous nasal surgery, sinusitis or respiratory infections in the previous 30 days, and asthma. Subjects were screened with the aid of a self-reported asthma questionnaire20 and diagnosed by medical examination, including pulmonary function testing when indicated by history. The healthy nonsmoker was defined as a subject who never smoked and had no diagnosis of acute or chronic diseases, with no use of medications except contraceptives, and had normal findings on physical examination. Self-reported nonsmokers with exhaled carbon monoxide (CO) levels > 9 parts per million (ppm) and/or cotinine levels > 10 ng/mL in nasal lavage fluid (NLF) were excluded from this study. Current smoking was defined according to the guidelines of the World Health Organization as subjects who have smoked ≥ 100 cigarettes and who currently smoke at least one cigarette a day.21 The Consolidated Standards of Reporting Trials (CONSORT) diagram is given in Figure 1.

Figure Jump LinkFigure 1. Consolidated Standards of Reporting Trials (CONSORT) diagram showing recruitment and subject study inclusion/exclusion.Grahic Jump Location

This study received approval by the institutional review board of the Faculdade de Medicina da Universidade de São Paulo Ethical Committee (CEP 147/13) and is registered at ClinicalTrials.gov (NCT01877291). All clinical investigations were conducted according to the Declaration of Helsinki of the World Medical Association.

Clinical Assessment and Pulmonary Function

All subjects were assessed between 7:00 am and 12:00 pm. Subjects completed a general health questionnaire. We also administered the 20-item Sino-Nasal Outcome Test (SNOT20)22 for symptoms of airway discomfort. Heart rate, systolic and diastolic BP, respiratory rate, and pulse oxygen saturation were recorded. To measure exhaled CO, subjects were asked to orally exhale slowly from their total lung capacity through a Micro CO analyzer (Cardinal Health U.K.232 Ltd) over 15 to 20 s.

Pulmonary function was measured using the Koko Legend spirometer (nSpire Health Inc) and using the American Thoracic Society/European Respiratory Society Task Force guidelines23,24 to determine FEV1 and FVC. The percentages of predicted spirometry values were calculated from published Brazilian population data.25

Nasal MCC

We evaluated the nasal MCC by measuring nasal saccharine transit time (STT).26 The subject was asked to avoid alcohol, tea, and coffee for 6 h and to eat or drink nothing for 2 h before the measurements. We first confirmed the subject’s ability to taste saccharine by placing a small amount directly on the tongue. The STT assessment was performed in a quiet room at a temperature of 21°C to 22°C and relative humidity of 63% to 71%. Subjects sat in a chair and were asked to maintain regular breathing and to avoid deep breathing, coughing, sneezing, sniffing, or talking during STT measurements. Saccharine particles (2.5 mg) were deposited 2 cm from the anterior end of the inferior nasal turbinate of the nonobstructed nostril, and the timer was stopped at the first perception of sweet taste. The maximum delay between the deposition and perception was set at 60 min for nondetection.

Nasal Mucus Collection and Mucus Surface Contact Angle

The subject extended his or her neck approximately 30°, and a mucus sample was collected with the aid of a soft brush in the opposite nostril to that used for the STT. Mucus samples were kept in coded, sealed plastic containers and immediately stored at −80°C until analyzed. All samples were analyzed for mucus contact angle.26,27 The contact angle measures the wettability of solid planar surface by a liquid and is measured at the liquid-air-solid interface. Surfaces that are very poorly wettable (like Teflon) are considered to be less “sticky.” Mucus that has a larger contact angle is more difficult to clear by coughing or sneezing.7,28 A 25-μL drop of mucus was dropped on a glass slide that had previously been treated with a sulfochromic solution to remove electrical charges and then washed with deionized water. The mucus was allowed to stabilize for 5 min, and the image was captured with the aid of a stereomicroscope (Stemi 2000C; Carl Zeiss) connected to a camera (Axiocam HSC; Carl Zeiss AG). The contact angle was measured using an image analysis program (Interactive AxionVison 4.7; Carl Zeiss AG).27

Exhaled Breath Condensate Collection and pH Analysis

It is difficult to noninvasively obtain specimens from the lower airways to assess inflammation. The exhaled breath condensate (EBC) provides information from the lower to the upper airways and can be repeatedly performed.29 For the EBC sample collection, the subject rinsed his or her mouth with distilled water and was instructed to swallow saliva and to hold a slight head extension (approximately 15°). The EBC sample was collected over 15 to 20 min of quiet regular tidal breathing through a mouthpiece that was connected to a collector device with dry ice (−78.5°C). The pH measurement was immediately performed in EBC samples. Approximately 0.5 mL of EBC was deaerated for 15 min with a 350 mL/min flow of ultrapure (99.9%) argon gas (Gama Gases Ltd). The pH was determined with the aid of a microelectrode and a pH meter (827 pH Laboratory; Metrohm Ltd). The pH meter was calibrated before each measurement using pH solutions 4, 7, and 9. The sample used for pH determination was discarded.

NLF Collection for Total and Differential Cell Counts and Cytokine Analysis

The subject performed a 30° neck extension in a sitting position. Five milliliters of 0.9% saline solution at room temperature was instilled into each nostril, and subjects were instructed to hold their breath without swallowing for 10 s.30 The subject expelled the NLF into a sterile plastic tube by bending the head forward and gently blowing both nostrils. NLF samples were centrifuged (10 min, 300g, 5°C); the supernatant was separated from the pellet and immediately transferred to sterile, coded polypropylene tubes and stored at –70°C for cytokine and cotinine levels determinations.

The pellet was suspended in 1 mL of phosphate-buffered saline for total cell counting in the Neubauer chamber (sample of 20 μL; × 400). Samples were centrifuged at 96g, 25°C, and 6 min for cytospin. The slide was stained with May-Grünwald-Giemsa, and the number of neutrophils, macrophages, lymphocytes, eosinophils, ciliated cells, and goblet cells were counted via microscopy (sample of 100 μL; × 1,000).30

The concentrations of tumor necrosis factor (TNF)-α, IL-4, IL-6, IL-8, IL-10, and myeloperoxidase (MPO) were determined by using High Sensitivity Human Cytokine Immunoassay HSCYTO-60SK and HCVD1-67AK Human Cardiovascular Disease Panel (Millipore), respectively. Reported detection limits were 0.106 pg/mL for TNF-α, 10 pg/mL for IL-4, 0.039 pg/mL for IL-6, 3.5 pg/mL for IL-8, 0.5 pg/mL for IL-10, and 0.062 ng/mL for MPO. The standard curve fit was between 0 pg/mL and 32 pg/mL for TNF-α, between 31.2 pg/mL and 2,000 pg/mL for IL-4, between 0 pg/mL and 10 pg/mL for IL-6, between 0 pg/mL and 2,000 pg/mL for IL-8, between 0 pg/mL and 50 pg/mL for IL-10, and between 0.156 ng/mL and 10 ng/mL for MPO. To determine cotinine levels in NLF, quantitative enzyme immunoassay (high-sensitivity cotinine immunoassay; DRG International, Inc) was used. The lower detection limit was 0.1, and the standard curve was fit between 0 ng/mL and 50 ng/mL.

Statistical Analysis

Data with normal distribution were expressed as mean ± SD. Nonparametric variables were expressed as median and interquartile range. Comparisons between young nonsmokers and smokers (< 2.5 pack-year history and those with ≥ 2.5 pack-year history) were performed by one-way analysis of variance with Bonferroni corrections or by Kruskal-Wallis test when appropriate. The χ2 test was used for analysis of sex and the SNOT20 results by groups. Correlations between variables were analyzed by using Pearson or Spearman coefficient correlation when appropriate. Multiple linear regression was performed to analyze EBC pH and adjusted for age and smoking history (pack-years). The difference was considered significant if P < .05.

Study Population

Seventy-two young subjects entered into the study: 32 nonsmokers and 40 smokers. Smokers were divided into two subgroups by smoking history: < 2.5 pack-years (n = 20) and ≥ 2.5 pack-years (n = 20). Smokers with ≥ 2.5 pack-year history were older than nonsmokers and smokers with < 2.5 pack-year history (Table 1). Smokers with ≥ 2.5 pack-year history had a greater BMI, increased heart rate, and increased systolic BP compared with nonsmokers (Table 1). The exhaled CO and cotinine levels in NLF confirmed the nonsmoking status (< 10 ppm and < 10 ng/mL, respectively) in all healthy nonsmokers. Smokers had higher exhaled CO and cotinine levels in NLF compared with nonsmokers (Table 1). There was a significant correlation between exhaled CO and cotinine levels (r = 0.58, P < .001), between pack-year history and exhaled CO level (r = 0.47, P < .001), and between pack-year history and cotinine level (r = 0.44, P < .001). No significant differences were found in FEV1 and FVC between smokers and nonsmokers.

Table Graphic Jump Location
Table 1 —Demographic Data for Healthy Young Adult Nonsmokers, Early Smokers With < 2.5 Pack-Y History and Smokers With ≥ 2.5 Pack-Y History

Data given as mean (SD) unless otherwise indicated. ANOVA = analysis of variance; bpm = beats per min; CO = carbon monoxide; IQR = interquartile range; NA = not applicable; ppm = parts per million.

a 

vs ≥ 2.5 pack-y smoking history.

b 

vs nonsmokers.

c 

χ2 test.

d 

Mann-Whitney test.

e 

Kruskal-Wallis test.

Reports of physical activity (at least twice weekly and 40 min each) were greater in smokers with < 2.5 pack-year history compared with smokers with ≥ 2.5 pack-year history (100% and 65% of the subjects, respectively; P = .012), but were similar to nonsmokers (84%). Those that performed physical activity had a lower heart rate compared with the sedentary subjects (n = 60; 65 ± 11 beats per minute [bpm] vs n = 12; 81 ± 12 bpm, respectively; P < .001).

There were no significant differences in the total score of SNOT20 between nonsmokers (0.37 ± 0.34), smokers with < 2.5 pack-year history (0.36 ± 0.33), and smokers with ≥ 2.5 pack-year history (0.63 ± 0.64; P = .140). However, smokers with ≥ 2.5 pack-year history complained more about cough (P = .05) and postnasal discharge (P = .016) than nonsmokers and smokers with < 2.5 pack-year history. Rhinitis is very prevalent in smokers, and the asthma screening questionnaire provided information about self-reported symptoms of rhinitis. The prevalence was 7.5% in nonsmokers (three of 40 subjects), 15% in light smokers (three of 20 subjects), and 20% smokers with ≥ 2.5 pack-year history (four of 20 subjects). No subject had clinical symptoms of rhinitis at the time of study inclusion.

Both subgroups of smokers had faster mean STT values (ie, a shorter time of 5.9 ± 3.1 min) than nonsmokers (7.7 ± 4.1 min, P = .033) (Fig 2). Mucus surface contact angle was similar between young smokers and nonsmokers, (40° ± 9° and 39° ± 6°, respectively; P = .803).

Figure Jump LinkFigure 2. The saccharine transit test and mucus contact angle comparing healthy young nonsmokers to early smokers with < 2.5 pack-y history and smokers with ≥ 2.5 pack-y history.Grahic Jump Location

The total number of cells in the NLF was increased in young smokers with more macrophages, ciliated cells, and goblet cells (Table 2). There were no significant differences in IL-4, IL-6, IL-8, IL-10, or TNF-α concentrations in NLF between groups. However, MPO concentration was increased in the NLF of light smokers (< 2.5 pack-year history).

Table Graphic Jump Location
Table 2 —Total Number of Cells and Differential Cellularity and Cytokines in Nasal Lavage Fluid of Healthy Young Adult Nonsmokers, Early Smokers With < 2.5 Pack-Y History, and Smokers With ≥ 2.5 Pack-Y History

Data given as median (IQR) unless otherwise indicated. MPO = myeloperoxidase; TNF-α = tumor necrosis factor-α. See Table 1 legend for expansion of other abbreviations.

a 

vs nonsmokers.

b 

Kruskal-Wallis test.

c 

vs ≥ 2.5 pack-y smoking history.

EBC pH was lower in smokers with ≥ 2.5 pack-year history compared with smokers who had < 2.5 pack-year history or nonsmokers (7.65 ± 0.42, 7.83 ± 0.26, and 7.90 ± 0.21, respectively) (Fig 3A). There was also a significant correlation between EBC pH and pack-year history (r = −0.47, P = .037), as well as between EBC pH and cotinine level (r = −0.51, P = .017). Linear regression analysis confirmed a significant dose-dependent effect of smoking in decreasing EBC pH (Fig 3B).

Figure Jump LinkFigure 3. A, EBC pH comparing healthy young nonsmokers to early smokers with < 2.5 pack-y history and smokers with ≥ 2.5 pack-y history. B, Linear regression between EBC pH and pack-y smoking history. EBC = exhaled breath condensate.Grahic Jump Location

We studied the effects of tobacco smoking in the upper airways of young adult smokers. These young smokers were asymptomatic; however, they showed early alterations in the upper airways. Specifically, these were faster nasal MCC, increased number of nasal cells (macrophages, ciliated cells, and goblet cells), increased concentrations of MPO, and decreased EBC pH. We demonstrated that these changes are dependent on the smoking history; more specifically, in the group studied, EBC pH decreased by 0.05 for each pack-year of smoking.

The EBC and NLF can be used to assess inflammatory biomarkers in the airway lining.3133 The airway EBC pH is usually about 8.2. A decrease in EBC pH may reflect acidification of the epithelial lining fluid, associated with an inflammatory response of the airways to the products of tobacco smoking and to an increase in the dissociation constant of the ammonium or bicarbonate buffer in the lining fluid.34,35 Smokers without signs of airway disease had similar EBC pH (approximately 8.17 vs approximately 8.16 in nonsmokers).35 However, subjects with > 10 pack-years of smoking had decreased EBC pH of about 7.40, and this is similar to stable and sick patients with COPD (pH 7.36 and 7.05, respectively). We also showed that tobacco smoking history is significantly associated with decreases in EBC pH.

We have reported STT results to be reproducible and we have used this technique to assess nasal MCC in different clinical conditions for > 10 years.26,36 Although the STT requires no sophisticated instruments, it can be challenging to obtain reproducible data, as placement of the saccharine requires skill and measurement requires a significant amount of patient coaching and cooperation. This could be considered a potential limitation to interpreting these results. Significant prolongation in STT (35%-120%) has been reported in long-term smokers,8,9,36,37 as well as no significant differences on STT between smokers and nonsmokers,38,39 and faster nasal ciliary beat frequency and transport in occasional smokers.18 In most of these studies, smokers were older, and morbidities were not always stated.26 In the present study, we showed faster STT in young “healthy” smokers than healthy nonsmokers. We speculate that young subjects who are early or light smokers may have a protective increase in ciliary beat frequency and transport in response to cigarette smoking. This may be due, in part, to persistence of this protective response. Experimental data in nasal ciliated cells obtained by brushing or biopsies showed increased ciliary beating directly induced by cigarette smoke extract40 or by tobacco smoking-induced inflammation.18

Smokers can have chronic cough and mucus retention with abnormalities in mucus hydration and properties.7 Mucus wettability (contact angle) is determined by surface forces, and increased surface adherence of mucus to the epithelium will decrease the effectiveness of cough.28 We found no differences in the mucus contact angle between smokers and nonsmokers. This is consistent with a previous study in young asymptomatic smokers that showed the same mucus cough clearability as in the mucus of nonsmokers.7

Nasal and sinus inflammation are common in patients with COPD.19 We used NLF to obtain concentrations of cytokines that might be important in initiating and sustaining airway inflammation.41,42 Consistent with our clearance data and with data demonstrating that most long-term smokers never develop COPD,14,15 we observed no differences in NLF cytokines between young smokers and nonsmokers except for an increase in MPO concentration, which may be the result of increased cells. Macrophages have been implicated in the pathogenesis of COPD.4345 Macrophages can be differentiated into proinflammatory phenotypes (M1 type) with release of MPO that may be an early marker of increasing inflammation in smokers without severe airway symptoms,46 and promotion of T helper cell 1 immunity.45 The epithelial cells may also have an important role in modulation of the inflammatory process, as they are a common source of IL-10 and other cytokines.47

In people who develop COPD, there is a relationship between smoking history and pulmonary function decrement. Approximately one-quarter of smokers develop spirometry changes diagnostic of COPD.14,15 In the present study, asymptomatic smokers showed no changes in pulmonary function, probably because spirometry is unlikely to detect early physiologic changes in the airways.

Our study design did not allow us to directly investigate smoke exposure effects in the lungs. We detected that airway EBC pH was decreased (ie, more acidic) in the young adult smokers, consistent with concomitant airway inflammation and correlated with smoking history. It is possible that smoking almost always induces a degree of inflammation, although it is unclear if this is dose dependent. The lower airway EBC pH decrease in these young smokers contrasted with the lack of changes in spirometry, suggesting that as in the nasal fluid, there may be inflammation without sufficient airflow limitation to produce COPD.

In conclusion, young adult smokers have functional and inflammatory changes in the nasal and lower airways and these correlate with smoking history. However, in these young smokers, smoking history was not associated with pulmonary function decline, probably because it is unlikely that spirometry detects early physiologic changes in the airways.

Author contributions: Dr Nakagawa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Ms Nicola: contributed to the data collection and analysis and writing of the manuscript and served as principal author.

Dr H. B. Carvalho: contributed to the statistical analysis, results analysis, discussion, and writing of the manuscript.

Ms Yoshida: contributed to the data collection, results analysis, discussion, and writing of the manuscript.

Dr Anjos: contributed to the data collection, results analysis, and writing of the manuscript.

Dr Nakao: contributed to the data collection and writing of the manuscript.

Dr Santos: contributed to the study design, data collection, results analysis, discussion, and writing of the manuscript.

Dr Cardozo: contributed to the results analysis and writing of the manuscript.

Dr V. M. Carvalho: contributed to the results analysis and writing of the manuscript.

Dr Pinto: contributed to the results analysis and writing of the manuscript.

Dr Farsky: contributed to the study design, results analysis, discussion, and writing of the manuscript.

Dr Saldiva: contributed to the results analysis and writing of the manuscript.

Dr Rubin: contributed to the results analysis, discussion, and writing of the manuscript.

Dr Nakagawa: contributed to the study design, data collection, statistical analysis, results analysis, discussion, and writing of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsor had no role in study design, data collection, data analysis, data interpretation, or preparation of the manuscript.

Other contributions: The authors would like to thank Matheus Cavalcante Sá, BSc, for helping with recruitment.

bpm

beats per minute

CO

carbon monoxide

EBC

exhaled breath condensate

MCC

mucociliary clearance

MPO

myeloperoxidase

NLF

nasal lavage fluid

ppm

parts per million

SNOT20

20-item Sino-Nasal Outcome Test

STT

saccharine transit time

TNF-α

tumor necrosis factor α

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de Oliveira-Maul JP, de Carvalho HB, Miyuki Goto DM, et al. Aging, diabetes, and hypertension are associated with decreased nasal mucociliary clearance. Chest. 2013;143(4):1091-1097. [CrossRef] [PubMed]
 
Daviskas E, Anderson SD, Jaques A, Charlton B. Inhaled mannitol improves the hydration and surface properties of sputum in patients with cystic fibrosis. Chest. 2010;137(4):861-868. [CrossRef] [PubMed]
 
Albers GM, Tomkiewicz RP, May MK, Ramirez OE, Rubin BK. Ring distraction technique for measuring surface tension of sputum: relationship to sputum clearability. J Appl Physiol (1985). 1996;81(6):2690-2695. [PubMed]
 
Davis MD, Hunt J. Exhaled breath condensate pH assays. Immunol Allergy Clin North Am. 2012;32(3):377-386. [CrossRef] [PubMed]
 
Belda J, Parameswaran K, Keith PK, Hargreave FE. Repeatability and validity of cell and fluid-phase measurements in nasal fluid: a comparison of two methods of nasal lavage. Clin Exp Allergy. 2001;31(7):1111-1115. [CrossRef] [PubMed]
 
Tanou K, Koutsokera A, Kiropoulos TS, et al. Inflammatory and oxidative stress biomarkers in allergic rhinitis: the effects of smoking. Clin Exper Allergy. 2009;39(3):345-53.
 
Ghafouri B, Ståhlbom B, Tagesson C, Lindahl M. Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteomics. 2002;2(1):112-120. [CrossRef] [PubMed]
 
Papaioannou AI, Koutsokera A, Tanou K, et al. The acute effect of smoking in healthy and asthmatic smokers. Eur J Clin Invest. 2010;40(2):103-109. [CrossRef] [PubMed]
 
Paget-Brown AO, Ngamtrakulpanit L, Smith A, et al. Normative data for pH of exhaled breath condensate. Chest. 2006;129(2):426-430. [CrossRef] [PubMed]
 
Koczulla AR, Noeske S, Herr C, et al. Acute and chronic effects of smoking on inflammation markers in exhaled breath condensate in current smokers. Respiration. 2010;79(1):61-67. [CrossRef] [PubMed]
 
Nakagawa NK, Franchini ML, Driusso P, de Oliveira LR, Saldiva PHN, Lorenzi-Filho G. Mucociliary clearance is impaired in acutely ill patients. Chest. 2005;128(4):2772-2777. [CrossRef] [PubMed]
 
Ramos EMC, De Toledo AC, Xavier RF, et al. Reversibility of impaired nasal mucociliary clearance in smokers following a smoking cessation programme. Respirology. 2011;16(5):849-855. [CrossRef] [PubMed]
 
Littlejohn MC, Stiernberg CM, Hokanson JA, Quinn FB Jr, Bailey BJ. The relationship between the nasal cycle and mucociliary clearance. Laryngoscope. 1992;102(2):117-120. [CrossRef] [PubMed]
 
Deniz M, Uslu C, Ogredik EA, Akduman D, Gursan SO. Nasal mucociliary clearance in total laryngectomized patients. Eur Arch Otorhinolaryngol. 2006;263(12):1099-1104. [CrossRef] [PubMed]
 
Navarrette CR, Sisson JH, Nance E, Allen-Gipson D, Hanes J, Wyatt TA. Particulate matter in cigarette smoke increases ciliary axoneme beating through mechanical stimulation. J Aerosol Med Pulm Drug Deliv. 2012;25(3):159-168. [CrossRef] [PubMed]
 
Amin K, Ekberg-Jansson A, Löfdahl C-G, Venge P. Relationship between inflammatory cells and structural changes in the lungs of asymptomatic and never smokers: a biopsy study. Thorax. 2003;58(2):135-142. [CrossRef] [PubMed]
 
Pelegrino NR, Tanni SE, Amaral RAF, Angeleli AYO, Correa C, Godoy I. Effects of active smoking on airway and systemic inflammation profiles in patients with chronic obstructive pulmonary disease. Am J Med Sci. 2013;345(6):440-445. [CrossRef] [PubMed]
 
Hogg JC, Chu F, Utokaparch S, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004;350(26):2645-2653. [CrossRef] [PubMed]
 
Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2008;8(3):183-192. [CrossRef] [PubMed]
 
Kunz LI, Lapperre TS, Snoeck-Stroband JB, et al; Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease Study Group. Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD. Respir Res. 2011;12:34. [CrossRef] [PubMed]
 
Andelid K, Bake B, Rak S, Lindén A, Rosengren A, Ekberg-Jansson A. Myeloperoxidase as a marker of increasing systemic inflammation in smokers without severe airway symptoms. Respir Med. 2007;101(5):888-895. [CrossRef] [PubMed]
 
Smith LA, Paszkiewicz GM, Hutson AD, Pauly JL. Inflammatory response of lung macrophages and epithelial cells to tobacco smoke: a literature review of ex vivo investigations. Immunol Res. 2010;46(1-3):94-126. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. Consolidated Standards of Reporting Trials (CONSORT) diagram showing recruitment and subject study inclusion/exclusion.Grahic Jump Location
Figure Jump LinkFigure 2. The saccharine transit test and mucus contact angle comparing healthy young nonsmokers to early smokers with < 2.5 pack-y history and smokers with ≥ 2.5 pack-y history.Grahic Jump Location
Figure Jump LinkFigure 3. A, EBC pH comparing healthy young nonsmokers to early smokers with < 2.5 pack-y history and smokers with ≥ 2.5 pack-y history. B, Linear regression between EBC pH and pack-y smoking history. EBC = exhaled breath condensate.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Demographic Data for Healthy Young Adult Nonsmokers, Early Smokers With < 2.5 Pack-Y History and Smokers With ≥ 2.5 Pack-Y History

Data given as mean (SD) unless otherwise indicated. ANOVA = analysis of variance; bpm = beats per min; CO = carbon monoxide; IQR = interquartile range; NA = not applicable; ppm = parts per million.

a 

vs ≥ 2.5 pack-y smoking history.

b 

vs nonsmokers.

c 

χ2 test.

d 

Mann-Whitney test.

e 

Kruskal-Wallis test.

Table Graphic Jump Location
Table 2 —Total Number of Cells and Differential Cellularity and Cytokines in Nasal Lavage Fluid of Healthy Young Adult Nonsmokers, Early Smokers With < 2.5 Pack-Y History, and Smokers With ≥ 2.5 Pack-Y History

Data given as median (IQR) unless otherwise indicated. MPO = myeloperoxidase; TNF-α = tumor necrosis factor-α. See Table 1 legend for expansion of other abbreviations.

a 

vs nonsmokers.

b 

Kruskal-Wallis test.

c 

vs ≥ 2.5 pack-y smoking history.

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de Oliveira-Maul JP, de Carvalho HB, Miyuki Goto DM, et al. Aging, diabetes, and hypertension are associated with decreased nasal mucociliary clearance. Chest. 2013;143(4):1091-1097. [CrossRef] [PubMed]
 
Daviskas E, Anderson SD, Jaques A, Charlton B. Inhaled mannitol improves the hydration and surface properties of sputum in patients with cystic fibrosis. Chest. 2010;137(4):861-868. [CrossRef] [PubMed]
 
Albers GM, Tomkiewicz RP, May MK, Ramirez OE, Rubin BK. Ring distraction technique for measuring surface tension of sputum: relationship to sputum clearability. J Appl Physiol (1985). 1996;81(6):2690-2695. [PubMed]
 
Davis MD, Hunt J. Exhaled breath condensate pH assays. Immunol Allergy Clin North Am. 2012;32(3):377-386. [CrossRef] [PubMed]
 
Belda J, Parameswaran K, Keith PK, Hargreave FE. Repeatability and validity of cell and fluid-phase measurements in nasal fluid: a comparison of two methods of nasal lavage. Clin Exp Allergy. 2001;31(7):1111-1115. [CrossRef] [PubMed]
 
Tanou K, Koutsokera A, Kiropoulos TS, et al. Inflammatory and oxidative stress biomarkers in allergic rhinitis: the effects of smoking. Clin Exper Allergy. 2009;39(3):345-53.
 
Ghafouri B, Ståhlbom B, Tagesson C, Lindahl M. Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteomics. 2002;2(1):112-120. [CrossRef] [PubMed]
 
Papaioannou AI, Koutsokera A, Tanou K, et al. The acute effect of smoking in healthy and asthmatic smokers. Eur J Clin Invest. 2010;40(2):103-109. [CrossRef] [PubMed]
 
Paget-Brown AO, Ngamtrakulpanit L, Smith A, et al. Normative data for pH of exhaled breath condensate. Chest. 2006;129(2):426-430. [CrossRef] [PubMed]
 
Koczulla AR, Noeske S, Herr C, et al. Acute and chronic effects of smoking on inflammation markers in exhaled breath condensate in current smokers. Respiration. 2010;79(1):61-67. [CrossRef] [PubMed]
 
Nakagawa NK, Franchini ML, Driusso P, de Oliveira LR, Saldiva PHN, Lorenzi-Filho G. Mucociliary clearance is impaired in acutely ill patients. Chest. 2005;128(4):2772-2777. [CrossRef] [PubMed]
 
Ramos EMC, De Toledo AC, Xavier RF, et al. Reversibility of impaired nasal mucociliary clearance in smokers following a smoking cessation programme. Respirology. 2011;16(5):849-855. [CrossRef] [PubMed]
 
Littlejohn MC, Stiernberg CM, Hokanson JA, Quinn FB Jr, Bailey BJ. The relationship between the nasal cycle and mucociliary clearance. Laryngoscope. 1992;102(2):117-120. [CrossRef] [PubMed]
 
Deniz M, Uslu C, Ogredik EA, Akduman D, Gursan SO. Nasal mucociliary clearance in total laryngectomized patients. Eur Arch Otorhinolaryngol. 2006;263(12):1099-1104. [CrossRef] [PubMed]
 
Navarrette CR, Sisson JH, Nance E, Allen-Gipson D, Hanes J, Wyatt TA. Particulate matter in cigarette smoke increases ciliary axoneme beating through mechanical stimulation. J Aerosol Med Pulm Drug Deliv. 2012;25(3):159-168. [CrossRef] [PubMed]
 
Amin K, Ekberg-Jansson A, Löfdahl C-G, Venge P. Relationship between inflammatory cells and structural changes in the lungs of asymptomatic and never smokers: a biopsy study. Thorax. 2003;58(2):135-142. [CrossRef] [PubMed]
 
Pelegrino NR, Tanni SE, Amaral RAF, Angeleli AYO, Correa C, Godoy I. Effects of active smoking on airway and systemic inflammation profiles in patients with chronic obstructive pulmonary disease. Am J Med Sci. 2013;345(6):440-445. [CrossRef] [PubMed]
 
Hogg JC, Chu F, Utokaparch S, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004;350(26):2645-2653. [CrossRef] [PubMed]
 
Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2008;8(3):183-192. [CrossRef] [PubMed]
 
Kunz LI, Lapperre TS, Snoeck-Stroband JB, et al; Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease Study Group. Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD. Respir Res. 2011;12:34. [CrossRef] [PubMed]
 
Andelid K, Bake B, Rak S, Lindén A, Rosengren A, Ekberg-Jansson A. Myeloperoxidase as a marker of increasing systemic inflammation in smokers without severe airway symptoms. Respir Med. 2007;101(5):888-895. [CrossRef] [PubMed]
 
Smith LA, Paszkiewicz GM, Hutson AD, Pauly JL. Inflammatory response of lung macrophages and epithelial cells to tobacco smoke: a literature review of ex vivo investigations. Immunol Res. 2010;46(1-3):94-126. [CrossRef] [PubMed]
 
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