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(Chest. 2006;130:1157-1164.)
© 2006 American College of Chest Physicians

Repeated Sputum Inductions Induce a Transient Neutrophilic and Eosinophilic Response*

Hester van der Vaart, MD; Dirkje S. Postma, MD, PhD; Wim Timens, MD, PhD; Henk F. Kauffman, MD, PhD; Machteld N. Hylkema, PhD and Nick H.T. ten Hacken, MD, PhD

* From the Departments of Pulmonology (Drs. van der Vaart, Postma, and ten Hacken), Pathology (Drs. Timens and Hylkema), and Allergology (Dr. Kauffman), University of Groningen, Groningen, the Netherlands.

Correspondence to: Nick H.T. ten Hacken, MD, PhD, University Medical Centre Groningen, Pulmonology, hanzeplein 1 Groningen 9713 RB, the Netherlands; e-mail: n.h.t.ten.hacken{at}int.umcg.nl

Abstract

Introduction: Sputum induction is a tool to monitor airway inflammation, yet it may induce by itself a neutrophilic response when repeated within 24 to 48 h. This limits its repeated use in clinical trials.

Objective: We aimed to investigate the induction and resolution of inflammation generated by repeated sputum inductions.

Subjects and design: Sixteen healthy intermittent smokers participated in a study on the short-term effects of smoking. The nonsmoking arm consisted of seven successive sputum inductions with increasing time intervals (3, 6, 12, 24, 48, and 96 h). Inflammatory cellular characteristics and different soluble mediators were investigated.

Measurements and results: The median percentage of sputum neutrophils increased significantly from baseline to 6 h (58.9% [range, 31.8 to 94.2%] to 83.2% [range, 26.7 to 98.3%], respectively). Surprisingly, the percentage of eosinophils also increased significantly from baseline to 6, 12, 24, and 48 h, as follows: 0.3% (range, 0.0 to 1.2%) to 1.7% (range, 0.0 to 15.5%), 2.2% (range, 0.5 to 12.5%), 1.2% (range, 0.0 to 4.8%), and 0.8% (range, 0.0 to 2.8%), respectively. Interleukin-8 increased significantly from baseline to 24 h (1,553 pg/mL [range, 462 to 8,192 pg/mL] to 2,178 pg/mL [range, 666 to 128,544 pg/mL]).

Conclusions: Repeated sputum inductions should preferably be avoided within 48 h. It induces not only a short-lived neutrophilic response but also a prolonged eosinophilic inflammatory response in healthy subjects, possibly by local changes in osmolarity, and subsequent epithelial and/or mast cell activation.

Key Words: eosinophils • inflammation • neutrophils • sputum induction

Sputum induction is a safe, noninvasive, and validated technique to assess airway inflammation in asthma and COPD patients, and healthy subjects.123 It is frequently used to monitor inflammation in clinical trials. A possible drawback for this application is that a neutrophilic response might occur when sputum inductions are being repeated within 8 or 24 h.45 This carryover effect thus limits the iterative use of sputum inductions. The minimum time interval between sputum inductions in order to obtain reproducible cellular counts was variable in different reports (ie, 24 and 48 h).678 Even less is known about the cumulative effect of more than two sputum inductions. Based on a review of the literature, the sputum induction working group of the European Respiratory Society9 stated that sputum inductions should not be repeated within 48 h to avoid carryover effects. In line with this, we used a crossover design (including no smoking) in a study on the subacute effects of smoking to correct for this carryover effect. Given the possible consequences of the repeated sputum inductions for its applicability in research, we have, as a separate study, used the control arm of the above study to investigate both the induction and resolution of the inflammatory response generated by repeated sputum inductions. To this aim, we analyzed the effects of seven repeated sputum inductions that were performed at increasing time intervals in 16 healthy volunteers.

Materials and Methods

Study Design
In a study of the subacute effects of smoking on inflammation,10 we designed a nonsmoking arm that consisted of seven sputum inductions separated by increasing time intervals (0, 3, 6, 12, 24, 48, and 96 h). Healthy subjects refrained from smoking for 9 days before and during the study period, which was verified by measuring the levels of exhaled carbon monoxide (CO) [< 6 ppm] and urinary cotinine (< 25 ng/mL).

Subjects
The 16 participants were > 40 years of age and had normal lung function, as follows: prebronchodilator FEV1/inspiratory vital capacity (IVC) ratio: women, > 89% predicted; men, > 88% predicted.11 All subjects had smoked more than one cigarette a month, but not daily, during the last 6 months. Subjects (1) with a history of asthma, allergic rhinitis, or allergic eczema; (2) with atopy, confirmed by a positive skin-prick test result; (3) with any current respiratory disease, symptoms of cough, or sputum production; (4) with respiratory tract infection within the preceding 8 weeks or a nasal infection within the preceding 4 weeks; and (5) had used aspirin, nonsteroidal antiinflammatory drugs, paracetamol, or antihistamines within the preceding 4 weeks were excluded from the study. The study was approved by the Medical Ethics Committee of the University Hospital Groningen, the Netherlands. Written informed consent was obtained from all subjects.

Pulmonary Function and Exhaled Carbon Monoxide
FEV1 and IVC were measured according to the guidelines of the European Respiratory Society11 using a pneumotachograph (Jaeger; Wurzberg, Germany). Exhaled CO levels were measured using an infrared CO analyzer (UNOR 6Nl Maihak AG; Hamburg, Germany).12

Sputum Induction
Sputum was induced according to the method of Rutgers et al13 with some modifications. Hypertonic sodium chloride aerosols (4.5% weight/V) were generated at room temperature by an ultrasonic nebulizer (Ultraneb 2000; DeVilbiss; Somerset, PA) with a calibrated particle size (mass median aerosol diameter, 4.5 µm) at maximum output (2.5 mL/min). Aerosols were administered through a 100-cm-long tube with an internal diameter of 22 mm and a two-way valve, and were inhaled via the mouth. Nose clips were used. Prior to sputum induction, 400 µg of salbutamol was administered for safety reasons, and FEV1 was measured. Hypertonic saline solution was nebulized during three periods of 5 min. After each inhalation period, or as soon as the subjects experienced cough, they were asked to blow their nose, rinse their throat with water, and cough their sputum into a plastic container. FEV1 was measured whenever a patient felt discomfort and after the completion of each inhalation maneuver. Salbutamol, 200 µg, was administered if FEV1 dropped > 20% from the post-salbutamol inhalation baseline value.

Sputum Processing
Whole sputum was processed within 120 min according to the modified method of Rutgers et al.13 In short, first the weight of the sputum samples was determined. An equal volume of 0.1% dithiothreitol (Sputolysin; Calbiochem; San Diego, CA) was added, and thereafter the sample was placed in a shaking water bath (at 37°C for 15 min). The homogenized sputum was filtered through a nylon gauze (pore-size, approximately 48 µm; Thompson; Scarborough, ON, Canada) to remove clumps, followed by centrifugation (at 450g for 10 min at 20°C). Differential cell counts of eosinophils, neutrophils, lymphocytes, macrophages, and epithelial and squamous cells were performed on May-Grünwald Giemsa-stained cytospins by a cytotechnician in a blinded fashion. For each sample, differential cell counts were performed on two cytospins (counting 2 x 300 nonsquamous cells) by the same observer. The mean data were used for statistical analysis. Differential leukocyte and cylindrical epithelial cell counts were expressed as a percentage of 600 nucleated cells, excluding squamous cells. A sputum sample was considered to be adequate when the percentage of squamous cells was < 80%. Absolute numbers of inflammatory cells per milliliter were calculated by multiplying the total number of sputum cells per milliliter by the differential count (it was assumed that 1 g of sputum equals 1 mL of sputum).

Immunocytology was performed to quantify the percentage of inducible nitric oxide synthase (iNOS)-positive macrophages. Cytospins were double-stained with a monoclonal antibody against CD68 (IgG1 isotype; Dako; Glostrup, Denmark) as a marker for macrophages and rabbit polyclonal antiserum against iNOS (Transduction Laboratories; Lexington, KY). The following soluble mediators were measured in sputum supernatant. NO2-/NO3- was measured using the Griess reaction, eosinophilic cationic protein (ECP) using a fluorenzyme immunoassay (UniCAP ECP; Pharmacia; Uppsala, Sweden). Interleukin (IL)-8 and leukotriene B4 (LTB4) were measured by a commercial enzyme-linked immunosorbent assay (IL-8: Sanquin; Amsterdam, the Netherlands; LTB4: Amersham Biosciences, Little Chalfont, UK). Matrix metalloproteinase (MMP)-9 was measured by gelatin zymography, and the tissue inhibitor of metalloproteinase-1 (TIMP-1) by enzyme-linked immunosorbent assay (R&D; Abingdon, UK). Neutrophil elastase activity was measured by chromogenic substrate assay (N-methoxysuccinyl-ala-ala-pro-val-p-nitoanilide; Sigma; Dorset, UK).14

Statistical Analysis
Clinical, cell, and mediator characteristics are presented as the median (range) [in tables] or as the median (interquartile range [IQR]) [in figures]. Outliers are presented between 1.5 and 3 times the IQR (in figures). Differences between the subsequent sputum inductions and baseline (t = 0) were tested with a Wilcoxon signed rank test. A p value of < 0.05 was considered to be statistically significant.

Results

Subjects
Clinical characteristics of the subjects are listed in Table 1 . All subjects successfully refrained from smoking for 9 days before and during the study period. All subjects tolerated the sputum induction procedure well, and no adverse events were reported during the study period.


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  		Table 1.. Subject Characteristics*

 
Sputum Quality
The quality characteristics of the sputum samples are shown in Table 2 . Thirty-four of the 112 sputum samples (30%) could not be evaluated for total and differential cell counts, due to the percentage of squamous cells being too high and/or low cell viability. Apart from a lower total number of sputum cells after a time interval of 6 h and a trend toward a lower viability after a time interval of 3 h compared to baseline (t = 0; p = 0.07), no differences were found in total cell count, cell viability, sputum weight, or squamous cell contamination at the different time points.


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  		Table 2.. Sputum Quality Characteristics*

 
Sputum Cells
The percentage and number of sputum cells are shown in Figures 1 and 2 , respectively. The percentage of neutrophils was significantly higher after a time interval of 6 h than at baseline (6 h, 83.2% [IQR, 26.7 to 98.3%]; baseline, 58.9% [IQR, 31.8 to 94.2%]). The number of neutrophils tended to be lower after a time interval of 6 h than at baseline (6 h, 375 x 103 cells/mL [IQR, 121 to 857 x 103 cells/mL]; baseline, 557 x 103 cells/mL [IQR, 138 to 1,757 x 103 cells/mL]; p = 0.051). Both the percentage of macrophages (3 h, 20.8% [IQR, 4.5 to 71.2%]; 6 h, 10.3% [IQR, 1.7 to 67.8%]; baseline, 38.5% [IQR, 4.2 to 64.0%]) and the number of macrophages (3 h, 119 x 103 cells/mL [IQR, 19.6 to 335 x 103 cells/mL]; 6 h, 42.2 x 103 cells/mL [IQR, 10.3 to 629 x 103 cells/mL]; baseline, 287 x 103 cells/mL [IQR, 24.4 to 2,217 x 103 cells/mL]) were significantly lower after time intervals of 3 and 6 h than at baseline. The percentage of eosinophils was significantly higher after time intervals of 6, 12, 24, and 48 h than at baseline (6 h, 1.7% [IQR, 0.0 to 15.5%]; 12 h, 2.2% [IQR, 0.5 to 12.5%]; 24 h, 1.2% [IQR, 0.0 to 4.8%]; 48 h, 0.8% [IQR, 0.0 to 2.8%]; baseline, 0.3% [IQR, 0.0 to 1.2%]). In addition, the number of eosinophils was significantly higher after a time interval of 12 h than at baseline (12 h, 25.0 x 103 cells/mL [interquartile, range, 2.2 to 115 x 103 cells/mL]; baseline, 1.9 x 103 cells/mL [IQR, 0.0 to 18.8 x 103 cells/mL]). Sputum induction had no significant effect on the percentage of lymphocytes, but the number of lymphocytes was significantly lower after time intervals of 3 and 6 h than at baseline (3 h, 2.0 x 103 cells/mL [IQR, 0.0 to 16.5 x 103 cells/mL]; 6 h, 0.7 x 103 cells/mL [IQR, 0.0 to 17.9 x 103 cells/mL]; baseline, 7.3 x 103 cells/mL [IQR, 0.0 to 38.8 x 103 cells/mL]). The percentage of iNOS-positive macrophages was significantly lower after a time interval of 24 h than at baseline (24 h, 54.2% [IQR, 20.0 to 81.7%]; baseline, 67.3% [IQR, 33.3 to 90.2%]).


Figure 1
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  		Figure 1.. Effect of repeated sputum inductions on the percentage of sputum cells at different time intervals. In each box plot, the bottom and the top of the box denote the 25th and 75th percentiles, respectively, the solid line is the median, and the brackets are outliers between 1.5 and 3 times the IQR. *p < 0.05 compared to baseline; ¶p = 0.06 compared to baseline.

 

Figure 2
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  		Figure 2.. Effect of repeated sputum inductions on the number of sputum cells at different time intervals. In each box plot, the bottom and the top of the box denote the 25th and 75th percentiles, respectively, the solid line is the median, and the brackets are outliers between 1.5 and 3 times the IQR. *p <0.05 compared to baseline; ¶p = 0.051 compared to baseline.

 
Inflammatory Mediators Sputum
Sputum ECP and IL-8 concentrations are shown in Figure 3 . The concentration of ECP tended to be lower after a time interval of 12 h than at baseline (12 h, 73.4 ng/mL [IQR, 8.0 to 804 ng/mL]; baseline, 75.1 ng/mL [IQR, 12.1 to 346 ng/mL; p = 0.06). The concentration of IL-8 was significantly higher after a time interval of 24 h than at baseline (2,178 pg/mL [IQR, 666 to 128,544 pg/mL]; baseline, 1,553 pg/mL [IQR, 462 to 8,192 pg/mL]) and tended to be lower after a time interval of 6 h than at baseline (6 h, 1,384 pg/mL [IQR, 379 to 12,966 pg/mL]; baseline, 1,553 pg/mL [IQR, 462 to 8,192 pg/mL; p = 0.06). Other sputum mediators are shown in Table 3 . Concentrations of nitrite/nitrate were significantly higher and concentrations of TIMP-1 were significantly lower after a time interval of 6 h than at baseline. Sputum induction had no effect on MMP-9 and LTB4 levels at any time point. Neutrophil elastase could be detected only in three sputum samples; therefore, no statistical analyses could be performed.


Figure 3
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  		Figure 3.. Effect of repeated sputum inductions on sputum IL-8 and ECP at different time intervals. In each box plot, the bottom and the top of the box denote the 25th and 75th percentiles, respectively, the solid line is the median, and the brackets are outliers between 1.5 and 3 times the IQR. *p < 0.05 compared to baseline; ¶p = 0.06 compared to baseline.

 

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  		Table 3.. Sputum Inflammatory Mediators*

 
Discussion

Sputum induction is a tool to monitor airway inflammation, yet some reports have shown a neutrophilic response when it is repeated within 24 to 48 h. This may limit its repeated use in clinical trials. We aimed to investigate both the induction and resolution of inflammation generated by repeated sputum inductions. The main result of our study was that repeated sputum inductions in healthy subjects generate a prolonged increase in the number of sputum eosinophils. This was not accompanied by an increased level of sputum ECP. In addition, repeated sputum inductions induced a short-lived increase in the percentage of sputum neutrophils and IL-8, and a short-lived decrease in the percentage and number of sputum macrophages. Altogether, the results of our study show that repeated sputum inductions should be avoided preferably, unless separated by > 48 h in time.

To our knowledge, it is a new finding that repeated sputum inductions induce an eosinophilic response in healthy subjects. In previous studies3415 in asthmatic patients, eosinophils increased after sputum inductions but were not reported to be significantly higher. This may be due to higher baseline levels of sputum eosinophils in asthmatic patients, and thus small changes in eosinophils are not significantly different. Another reason may be that our study especially focused on the effects of repeated sputum inductions at relatively short time intervals. Interestingly, the levels of sputum eosinophils remained elevated for a longer period of time (time interval, 48 h) than those in our study. We can only speculate on this differential effect. Apparently, the recruitment and activation of eosinophils and neutrophils by sputum inductions differ. In this context, eosinophils can be directly activated by hyperosmolar stimuli,16 while they also show increased sensitivity for mast cell mediators.17 Regarding ECP levels, Holz et al4 have shown that sputum ECP levels were increased in healthy subjects as well as in asthmatic subjects 24 h after the first sputum induction. In contrast, in our study sputum ECP levels tended to decrease; the reason for this discrepancy is unclear.

Repeated sputum inductions increased the percentage of neutrophils after a time interval of 6 h in healthy subjects in our study. This is in line with the results of two other studies45 showing increased percentages and numbers of sputum neutrophils in healthy subjects at 8 and 24 h. Several mechanisms have been proposed to explain the increase in neutrophils. First, hypertonic saline solution may induce local changes in osmolarity,18 which may subsequently result in the release of proinflammatory mediators from mast cells or epithelial cells.19202122 Second, frequent coughing during the sputum induction may irritate the mucosa and result in a neutrophilic influx into the airway lumen. Increased neutrophils in our sputum due to lipopolysaccharide contamination of the hypertonic saline solution23 is not very likely, since no lipopolysaccharide could be demonstrated in our inhalation fluid.

In contrast to the increase in the percentage of neutrophils, we observed a decrease in the number of neutrophils, which may be due to the significantly lower total sputum cell count at that time. Similarly, we observed a lower number and percentage of macrophages after time intervals of 3 and 6 h, respectively, which seems to represent a real decrease, as this is more than is to be expected based on a reduced total cell count. An explanation for this decrease may be that macrophage kinetics are rather slow. After a washout of macrophages by sputum induction, it may take > 6 h (up to at least 12 h) before local homeostasis is reached and again reflects the nonaffected inflammatory status. Furthermore, the percentage of iNOS-positive macrophages decreased as well after a time interval of 24 h. It has been shown previously24 that the level of exhaled nitric oxide decreases after a single sputum induction, which might be explained by the decrease in iNOS observed in our study.

With respect to mediators in sputum, we have found that repeated sputum inductions increased IL-8 levels in the sputum supernatant after a time interval of 24 h. No increase in neutrophils was seen at that time point, which makes it less likely that this cell is a source of IL-8. Hashimoto et al20 showed increased IL-8 expression when epithelial cells were exposed to a hyperosmolar challenge in vitro. Furthermore, IL-8 expression in the bronchial epithelium increased after lavage with an isotonic saline solution challenge in asthmatic subjects.25 We therefore assume that epithelial cells are the main source for IL-8 in our study.

No significant differences were observed for any marker between sputum inductions performed at time intervals of 48 and 96 h. Therefore, we confirm and extend the conclusion of the European Respiratory Society working group,9 showing that > 48 h is needed to avoid a carryover effect, even if several repeated sputum inductions have been performed.

Our sputa had a relatively high percentage of squamous cells (60.5% [IQR, 3.8 to 94.7%]) compared to percentages reported in other studies (means and medians varying between 1.2% and 53%).13526 This might be due to differences in the methods used for induction and processing of the sputum or due to our specific study group of healthy intermittent smokers. Many studies27 used selected sputum plugs, which are known to contain fewer squamous cells, for cytospin preparation. The relatively high percentage of squamous cell contamination might have lowered the actual concentration of the cytokines IL-8 and ECP,28 though various findings on the effects of squamous cells on the supernatant and cells have been published.329 In addition, sputa from our repeated inductions had relatively low viability (61.4% [IQR, 9.2 to 88.6%]) compared with other studies (medians and means varying between 70% and 88%).4630 In our study, the viability and the total cell count decreased after the time intervals of 3 and 6 h, respectively, compared to baseline, which may be due to the short time interval between the sputum inductions, as has been shown by Richter et al.31 The low total cell count in our study may explain the decreased numbers of macrophages, neutrophils, and lymphocytes, but strengthens the observed influx of eosinophils.

Our time-dependent variations in sputum cells cannot easily be explained by a circadian variation. Blood leukocytes have a circadian rhythm with peak levels between 10:30 P.M. and 2.30 A.M.32 Only one study33 has been published investigating the circadian variation of inflammatory cells in sputum, showing higher eosinophils at 7:00 AM compared to 4:00 PM in asthmatic patients, which is not in line with our data. We therefore assume that a circadian rhythm does not explain our results.

Summarizing, our results show that repeated sputum inductions generate inflammatory changes. An interesting new observation is that repeated sputum inductions generate a prolonged eosinophilic inflammatory response. We can only speculate as to the mechanisms underlying this prolonged activation, but a role for mast cells seems attractive. Consistent with the literature, sputum induction induces a short-lived neutrophilic inflammatory response, possibly caused by local changes in osmolarity and subsequent epithelial cell and/or mast cell activation. Further, at the same early time point macrophages seem to have delayed kinetics, resulting in a slow restoration of normal levels after sputum induction. Altogether, it can be concluded that repeated sputum inductions should preferably be avoided, unless separated by > 48 h in time.

Acknowledgements

The authors thank M.A. Star-Kroezen, A.J. van der Laan-Boers, and E.M.D.H. Swierenga (Lung Function Department) for the many lung function measurements and sputum inductions they performed; I. Sloots, M. van der Toorn, H.A. Buivenga-Steketee, and J.A. Noordhoek (Laboratory of Pulmonology and Allergology) for all measurements in sputum and blood; M.D.W. Barentsen (Laboratory of Pathology) for the counting of the iNOS-positive macrophages on cytospins; and Drs. R.A. Uges and B. Greijdanus (Department of Pharmacy) for measurements of urinary cotinine levels.

Footnotes

Abbreviations: CO = carbon monoxide; ECP = eosinophilic cationic protein; IL = interleukin; iNOS = inducible nitric oxide synthase; IQR = interquartile range; IVC = inspiratory vital capacity; LTB4 = leukotriene B4; MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinase

Parts of this study were presented in a poster at the American Thoracic Society 2004 annual meeting.

This study was funded by AstraZeneca, Lund, Sweden.

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Received for publication January 10, 2006. Accepted for publication March 21, 2006.

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