Induced sputum in the investigation of airway inflammation of COPD

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1 Respiratory Medicine (2003) 97, Induced sputum in the investigation of airway inflammation of COPD M. Tsoumakidou, N. Tzanakis*, N.M. Siafakas Department of Thoracic Medicine, Medical School, University of Crete, P.O. Box 1352, Heraklion, Crete 71110, Greece KEYWORDS Sputum induction; COPD; Inflammation Summary During the last decade, the method of sputum induction (SI) has offered the opportunity to study inflammation in patients with chronic obstructive pulmonary disease (COPD). This paper reviews methodological aspects of SI and summarizes its uses in the research of inflammation in COPD, including sputum cellularity and soluble markers. SI is a relatively safe, reliable, and reproducible technique, used to investigate different aspects of airway inflammation. Although various methods of induction and processing have been proved safe and highly reproducible, a generally accepted method is needed. Sputum analysis has given evidence for increased numbers of macrophages and neutrophils in COPD patients compared to normal subjects. In some studies, increased numbers of eosinophils have been also reported. Changes in various mediators have been found in sputum supernatant of COPD patients (IL-8, LTB-4 and TNF-a). The clinical usefulness of the method in the followup of the disease has not been explored extensively. A number of observations in patients with different clinical characteristics could be proven useful in identifying patterns of inflammation associated with different prognosis. Finally, SI could also guide treatment; such as, sputum eosinophilia in COPD could predict response to inhaled corticosteroids. r 2003 Elsevier Ltd. All rights reserved. Introduction The inhalation of an aerosol of hypertonic saline to produce sputum was firstly used by Bickerman et al. in 1958, 1 in cytology of lung cancer. Later, induced sputum (IS) was used for clinical purposes to detect numerous pathogenic microorganisms. 2 4 At the beginning of 90s, sputum induction (SI) was introduced by Pin and colleagues as a method to study airway inflammation in asthma. 5 Thereafter, this method has been extensively used in the field of research of airway inflammation and it has considerably improved our understanding of *Corresponding author. Tel: þ ; fax: þ address: tzanakis@med.uoc.gr (N. Tzanakis). the most common pulmonary diseases, including asthma and chronic obstructive pulmonary disease (COPD). Methodology Sputum induction SI consists of inhaling an aerosol of saline produced from an ultrasonic nebulizer. Due to the relative invasiveness of this method, bronchodilation medication, oxygen supply and resuscitation equipment should be available. Although an experienced technician can conduct SI, a physician should supervise the procedure. The above limitations, along with the relative high cost of the nebulizer, probably limit the use of SI to specialized centers /03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi: /s (03)

2 864 M. Tsoumakidou et al. Disagreement exists between various investigators on how the procedure of SI should be done and on whether the entire sputum or just its plugs should be collected and analyzed. This creates confusion when data of sputum studies using different methodology are compared. The European Respiratory Society (ERS) has already set-up a Task Force in an attempt to standardize SI, sputum processing and analysis. 6,7 Two are the main methods to induce sputum. Iredal et al. 8 propose the inhalation of the same concentration of hypertonic saline (4.5%) for increasing time intervals, while Pin et al. 5 propose the inhalation for the same period of increasing concentrations of hypertonic saline (3%, 4%, 5%). One study has shown that hypertonic saline 3% is as successful in inducing sputum as hypertonic saline 3 5% given sequentially. 9 In our laboratory, we use the method of Pin et al. 5 with a success rate more than 95%, and without serious complications even in severe COPD patients Briefly, before the beginning of the procedure, the baseline FEV 1 is recorded and an inhalation of MDI salbutamol (200 mg) is given, followed by a spirometry 10 min later. It has been shown that b2 agonist pretreatment has no effect on sputum inflammatory percentages and ECP concentration in sputum, however it decreases histamine concentration. 9,14 Then SI is performed. The subject inhales hypertonic saline for maximum 10-min sessions. The concentration of the inhaled saline is consecutively increased in each session from 3% to 4% and to 5%. Always between sessions the inhalation procedure is interrupted and the subjects are asked to blow their nose, rinse their mouth and try to expectorate sputum into a sterilized box. By this way saliva contamination of the sample is minimized and percentage of squamous cells in the sample is decreased. 15 The best quality of samples is achieved by using controlled exhalation and coughing effort. Expectoration of secretions by sniffing or gargling should be avoided. This stage is very crucial to select samples representative of the airways secretions. Between inhalation sessions a spirometry is always performed to detect significant fall of FEV 1. The procedure is stopped when an adequate sputum sample is collected (approximately 1 g of plugs), or if troublesome symptoms have been presented or/ and if FEV 1 falls more than 20% of the baseline measure observed. It is not clear by which mechanism the inhalation of hypertonic saline produces sputum. An osmotic effect 16,17 or an increase in mucocilliary clearance 18,19 and/or stimulation of glandular secretions is the proposed explanation. Sputum processing The sputum should be processed within the next 30 min or no more than 2 h, with the sample always kept in ice. Numerous laboratories prefer to process the entire sputum, a method introduced by Fahy et al. 20 Others prefer to collect and analyze the more viscid proportions of the sputum (plugs), as described by Pin et al. 5 and modified by Popov et al. 9 Both procedures have been shown to be valid and reproducible, but the method of selected sputum-plugs provides higher cell viability and quality. 21,22 Briefly, after the selection and weighing of sputum plugs, dithiothreitol (DTT) is added, followed by Dulbecco s phosphate buffer saline (D-PBS). Then, the mixture is filtered and centrifuged. The supernatant is aspirated and frozen at 801C for analysis later. The cell pellet is resuspended with D-PBS plus 10% FCS (fetal calf serum). A total cell count (TCC) of the filtered sample is always performed, preferentially before centrifugation 23 and viability is tested by means of trypan blue exclusion method. If cell viability is less than 50% and/or squamous cell contamination is more than 20%, the sample cannot be used because results have been shown to be less reproducible. 24 Sputum cell suspensions are kept into eppendorf cups. Cytospins are also prepared. Both are kept at 801C for later processing. Differential cell counts (DCC) are usually performed on May-Grunwald Giemsa stained cytospins, excluding squamous epithelial cells. In the first step of sputum processing, DDT is added. DDT is a reducing agent destructing mucus glycoproteins and is used to liquefy and to homogenize sputum. However, DDT could destroy important cell surface markers (epitopes) and other free soluble molecules (mediators). It might also interfere with the capture antibodies of immunoassays. Various studies have shown the effect of DDT on cell viability, 25 on lymphocyte, eosinophilic, neutrophilic markers and on soluble mediators. 30,31 Consecutively, the DDT effect on the marker of interest should always be considered. Flow-cytometry Some cellular markers can be analyzed right away, if the sample is processed to flow cytometry. Briefly, sputum cells (approximately 1 million cells) are treated with albumin for 10 min and then with the antibodies chosen for 30 min at 371C. The mixture washed twice with D-PBS is ready for analysis. If treated with formaldehyde it can be analyzed within the next 1 2 days. In our

3 Analysis of induced sputum in COPD 865 laboratory samples prepared as described above are analyzed on an EPICS ELITE fluorescence activated flow-cytometer as follows. 10,11,13,32 The cells are tightly gated by volume and complexity on a forward (01) and side light scattering (901) mode. The appropriate control is always used for subtraction of the background. The percentage of one-, two- or three-color positive cells is measured and the mean channel value (MCV) as well as the relative fluorescence intensity (RFI) corresponding to the antigen density is estimated. The QC-Combo Kit (FCSC, San Juan, USA) is used for quantification of antibody binding. The number of positive cells in gated cell populations that are stained with the labeled antibody/antibodies is measured and expressed as percentage of gated cells. Day-to-day instruments calibration (amplification and compensation settings of the flow cytometer) is routinely followed. This method allows the simultaneous staining of cells with different markers and, therefore, can be used to analyze cellular subtypes or cellular activation. 33 However, until nowadays it cannot separate eosinophils from neutrophils with accuracy. 34 On the contrary, it is preferred for lymphocyte analysis in sputum samples. Sputum usually contains very low numbers of lymphocytes (1 2%). For this reason, we analyze approximately 1 million of sputum cells, among which there should be at least lymphocytes. Using flow-cytometry we have recently analyzed sputum T-lymphocytes subsets and intracellular expression of perforin in sputum CD8 þ T-lymphocytes. 10,11,13,32 Lymphocytes can be difficult to gate from other cells, unless gated by volume and complexity and by CD45 þ expression (pan leukocyte marker). 32 The use of anti-cd45 antibody (monoclonal mouse antihuman) is most crucial, because it allows the differentiation of epithelial cells from lymphocytes. Figures 1 and 2 clearly demonstrate the difference in the quality of data obtained with and without anti-cd45 antibody use. The following mouse anti-human monoclonal antibodies are successfully used in our laboratory for labeling sputum lymphocyte cells: anti-cd3, anti-cd4 and anti-cd8, anti-perforin, as well as unlabelled anti-humanperforin for intracellular perforin detection. The above antibodies are peridin chlorophyll protein (PerCP)-, phycoerythrin-cyanine (PCy-5), or FITCconjugated. Mouse anti-mouse isotype matched PerCP-, Pcy-5-, or FITC-conjugated immunoglobulins are, therefore, used as control antibodies. In general, following the above method we can obtain clear data in about 90% of our sputum samples. In the remaining 10% we fail to analyze sputum lymphocytes due to their extremely low FS Figure 1 Histogram of sputum cells from a COPD patient. The CD45 expression was not used for gating lymphocytes. Gating of lymphocytes is impossible. FS: forward scattering mode, SS: side light scattering mode. FS B Figure 2 Histogram of the same sputum cells, using the CD45 expression for gating lymphocytes. Gating of lymphocytes is possible (circle). FS: forward scattering mode, SS: side light scattering mode. number in some samples (o1%), or because of their destruction during sputum processing. Reproducibility There have been studies showing that sputum cell counts are highly reproducible both within investigator and between investigators, 35,36 with the greater repeatability when cell viability is high SS SS 64 64

4 866 M. Tsoumakidou et al. and squamous cell contamination low. 37 Highly reproducible results have been also reported in studies examining sputum samples from the same individual induced on different days. 35,36,38 41 However, other investigators showed that consecutive samples of IS might differ with respect to cellular composition. 42,43 Nightingale et al., have recently reported that repeated induced sputum in healthy volunteers within 8 or 24 h, showed considerable increases in neutrophil counts and percentages. 42 According to the authors this observation is attributed to an inflammatory response of the airways to the induction procedure. There is accumulating evidence, from studies in normal subjects as well as in asthmatics, that high saline concentration induces airway neutrophilia A high decrease in the time interval between two sputum samples may significantly influence the results Indeed different samples obtained at different inhalation periods differ with respect to cell differentiation and fluid phase markers. 44 Furthermore, differences are observed depending on the type and the severity of the disease. 46 These data have led to the hypothesis that repeated SI within a time scale of 40 min constitute of samples from different parts of bronchial tree. Some evidence suggests that the initial samples possibly represent the more central airways, while the last ones, the more peripheral ones. 44,45 If such a hypothesis is confirmed, then the duration of the induction procedure should be standardized and always mentioned in a study. Safety in COPD It is known that SI may provoke bronchoconstriction. Various mechanisms may be involved in the observed bronchoconstriction; the hypertonicity of the saline, the total inhaled amount, the rate of delivery. 9,43,47 Although several studies assess the safety of IS in asthma, there is little information concerning COPD. One study reports no side effects of the procedure in COPD patients, 35 while three others report clinically important dyspnea and/or fall in FEV 1 410% in a number of patients, ranging from 15% to 47%. 38,51,52 Patients whose airway obstruction is more reversible seem to be better protected by bronchodilator pretreatment. 51 These results indicate the necessity of monitoring spirometry of a COPD patient during the SI procedure in order to detect on time airway constriction. In our laboratory with any fall in FEV 1 between 10% and 20% of baseline we do not proceed to an increase in the concentration of saline, while if we detect a 20% fall, we stop the procedure. We have performed more than two hundred SI in moderate and severe COPD patients without significant clinical side effects In general, the risks from SI in patients with COPD are acceptable. Inflammatory indices in COPD obtained by sputum analysis Inflammatory cells Sputum analysis has shown that in the airway lumen of patients with COPD, the neutrophil is the predominant cell. Increased numbers of neutrophils have been found in sputum samples of stable COPD patients Moreover, a negative correlation has been reported between the percentage of neutrophils and FEV 1. 35,55,58 The neutrophilic inflammation partly explains the resistance of most COPD patients to steroid treatment, 59 though some investigators report a decrease in the neutrophilic counts after steroid treatment. 60,61 Another cell, which recently has gained much attention for its potential involvement in COPD pathogenesis, is the macrophage. Though sputum samples in COPD patients contain less macrophages than neutrophils, investigators have used sputum analysis to study the role of macrophage metalloproteinases in tissue remodeling and airway obstruction Sputum analysis also involves the eosinophil in COPD pathogenesis. Saetta et al. report increased sputum eosinophil numbers on COPD exacerbations. 65 Other investigators do not confirm this finding. 66 However, many studies have demonstrated sputum eosinophilia (sputum eosinophilsx3%) in COPD patients under stable conditions, as well as an inverse correlation between the number of sputum eosinophils and FEV 1 value. 67,68 This is also a clinically relevant observation since COPD patients with sputum eosinophilia respond better to treatment with inhaled steroids. 69,70 Controversy exists on whether eosinophils in COPD are activated or not. 67,68 Although sputum samples are rich in neutrophils, eosinophils and macrophages, they contain relatively small numbers of lymphocytes. These cells have been extensively studied in bronchial biopsy specimens and bronchoalveolar lavage fluid (BALF) from patients with COPD, but not in sputum. BALF and biopsy studies showed an increase in the total numbers of T-lymphocytes, with the predominance in the CD8 þ ve cytotoxic T-cells In

5 Analysis of induced sputum in COPD 867 our laboratory, lymphocytes in sputum and their subpopulations were investigated by using flowcytometry, or by isolating lymphocytes from sputum samples and proceeding to immunocytochemical analysis. By using these methods, we achieved to measure T-lymphocyte subpopulations. A decrease in the CD4/CD8 ratio, as well as a decrease in the Tc1/Tc2 ratio (Tc1 and Tc2 cells are subpopulations of CD8 þ ve cells) in COPD smokers compared to healthy smokers, was observed. 10,11 Moreover, an increase in perforin, which is released by CD8 cells and causes cytolysis, was found. 13 Inflammatory mediators The inflammatory process of COPD is complex. It involves not only inflammatory cells, but also certain mediators, which interact with each other and with the relevant cells. Nowadays numerous inflammatory mediators are being measured in the fluid phase of sputum. The precision and validity of these measurements greatly depends on the assay, on the mediator measured, on the nature of sputum itself, as well as on the effect of sputum processing on both the mediator measured or the antibodies used. Unfortunately, the standard mediator used in an immunoassay might differ from the endogenous mediator and from the standard mediator in another assay, making comparisons between different assays invalid. An effort is being done to standardize different assays, so that valid comparisons can be done. 74 Despite the above considerations, it is acceptable to measure mediators in sputum supernatant, as long as attention is given to the method of measurement and results are carefully interpreted. Keatings et al., have shown that sputum samples from COPD patients are characterized by an increase in eosinophilic cationic protein (ECP) and eosinophil peroxidase (EPO), which are the major eosinophilic granule proteins, as well as in myeloperoxidase (MPO) and human neutrophil lipocalin (HNL), which are both neutrophilic granule proteins. 59 These findings suggest that neutrophils, as well as eosinophils, are activated in COPD patients. Moreover, leukotriene B-4 (LTB-4), which is chemoattractant to neutrophils, has been recently reported increased in the sputum of patients with COPD. 75 Tumor necrosis factor-a (TNF-a), a proinflammatory cytokine known to up regulate adhesion molecules, and interleukin-8 (IL-8), a potent neutrophil attractant, both have been reported to be elevated. 55 The levels of both chemoattractants of neutrophils (IL-8 and LTB-4) correlate positively to MPO and neutrophi elastase (NE). 76 In the same direction, lower sputum levels of IL-10, a cytokine known to down regulate inflammatory responses, have been found in COPD patients. 77 A lower level of IL-10 positive cells was also reported in the same study. 77 Sputum analysis makes possible the study of mediators involved in the pathogenesis of COPD exacerbations. Increased levels of IL-8 and TNF-a have been observed during an acute exacerbation of COPD, independently of airway infection. 78 According to the results of a study by Bhowmik et al., high IL-6 and IL-8 levels in stable COPD are related to high frequency of exacerbations. 66 In the same study, the analysis of sputum samples obtained on an exacerbation revealed high levels of IL-6, a cytokine known to promote immunoglobulin production. Interestingly, these levels related to the presence of a common cold and to the eosinophil and lymphocyte numbers. In contrast, Gompertz et al., in a recent study reported no relationship between baseline levels of numerous mediators such as IL-8, MPO, leucotriene B-4 (LTB- 4) and secretory leucoproteinase inhibitor (SLPI), and frequency of exacerbations. 79 Finally, endothelin-1 (ET-1), a bronchoconstrictor peptide, has been reported to be increased in sputum samples of COPD patients during an exacerbation. 80 In conclusion, in either stable or exacerbated COPD, several inflammatory mediators are measured in induced sputum. This indicates that a complex interaction between cells and mediators takes part in the progressive obstructive deterioration in the small airways and in the destruction of lung parenchyma observed in COPD patients. 54 Comparison of induced sputum with spontaneous sputum, BAL and bronchial biopsies Spontaneous sputum, IS, BALF, bronchial biopsies and surgical specimens are the common methods to study lung inflammation. Surgical specimens exceed all others in that they offer the chance of studying structure, as well as inflammation of the entire lung, airways, arteries and parenchyma. Still, difficulties in their acquisition and analysis have made them the least used method. Spontaneous sputum samples, though completely safe, are also rarely studied because they contain less viable cells than IS samples. 52,81 Interestingly, no difference has been found in inflammatory cells 41,52 and IL-8 52 between spontaneous and IS. Pizzichini et al., in a study in asthmatic patients showed that a viability of the spontaneous sample higher than 50%

6 868 M. Tsoumakidou et al. predicts the agreement between fluid phase markers. 41 Possibly, in cases when SI cannot be safely applied, like in COPD exacerbations, spontaneous sputum samples could be an acceptable alternative, but this has to be proven. Although IS may show the same inflammatory profile with spontaneous sputum, differences are observed compared to BALF or biopsies. Studies in COPD patients have shown that sputum, BALF and bronchial biopsies represent different lung compartments. 57,82 Sputum mainly originates from the large airways, bronchial biopsies from the large or small airways depending on the site chosen and BALF from the bronchioles and alveoli. In COPD sputum consists mainly of neutrophils, while it has less macrophages and even less lymphocytes. BALF, on the other hand, consists mainly of macrophages, with less neutrophils and lymphocytes. Interestingly, sputum has the advantage of a more concentrated sample and contains more cells, as well as more fluid-phase markers than BALF. 83 In biopsies, the lymphocyte is the predominant cell, 82 while there is no great difference in eosinophils between sputum, BALF and biopsy specimens. 82 Same observations have been also made in respective studies in asthmatic subjects Clinical applications As shown earlier, sputum analysis gives evidence for a close association between airway inflammation and COPD pathogenesis. In this light SI could be used in clinical practice for the assessment of airway inflammation in COPD. Moreover, it is possible that different sputum inflammatory profiles are connected to different disease phenotypes. For example, sputum eosinophilia has been found characteristic of patients with steroid response and sputum analysis might serve as a screening test before deciding on long-term corticosteroid treatment in COPD. Although SI has much potential to develop as a clinical tool (it is a simple, safe and well-tolerated method) convincing data for any usefulness in clinical practice is lacking. Once this method is introduced for routine assessment of COPD patients the issue of cost effectiveness has to be considered as well. Conclusion In conclusion, SI is a relatively reliable and safe method to study airway inflammation in COPD. These unique characteristics make it a useful tool in the investigation of the complex inflammatory processes of COPD. So far, it has showed interactions between inflammatory cells and mediators, which are related to physiological consequences of obstruction. On the other hand, sputum analysis could be used in clinical practice as a monitoring or screening test. Considering the importance of the above method in the research and its possible clinical applications, a further development of the technique is expected. This includes standardization and introduction of methods to investigate new markers of inflammation. The use of flowcytometry could also enhance the applications of SI. Acknowledgements The writers thank Koutala Heleni for her collaboration on flow-cytometric analyses. References 1. Bickerman HA, Sproul EE, Barach AL. An aerosol method of producing bronchial secretions in human subjects: a clinical technique for the detection of lung cancer. Dis Chest 1958;33: Bandyopadhyay T, Gerardi DA, Metersky ML. A comparison of induced and expectorated sputum for the microbiological diagnosis of community acquired pneumonia. Respiration 2000;67(2): Leigh TR, Parsons P, Hume C, Husain OA, Gazzard B, Collins JV. Sputum induction for diagnosis of Pneumocystis carinii pneumonia. Lancet 1989;2(8656): Larson JL, Ridzon R, Hannan MM. Sputum induction versus fiberoptic bronchoscopy in the diagnosis of tuberculosis. Am J Respir Crit Care Med 2001;163(5): Pin I, Gibson PG, Kolendowicz R, Girgis-Gabardo A, Denburg JA, Hargreave FE, et al. Use of induced sputum cell counts to investigate airway inflammation in asthma. Thorax 1992;47(1): Djukanovic R, Sterk PJ, Fahy JV, Hargreave FE. Standardised methodology of sputum induction and processing. Eur Respir J Suppl 2002;37:1s 2s. 7. Vignola AM, Rennar SI, Hargreave FE, Fah JV, Bonsignore MR, Djukanovic R, et al. Standardised methodology of sputum induction and processing. Future directions. Eur Respir J Suppl 2002;37:51s 5s. 8. Iredale MJ, Wanklyn SA, Phillips IP, Krausz T, Ind PW. Noninvasive assessment of bronchial inflammation in asthma: no correlation between eosinophilia of induced sputum and bronchial responsiveness to inhaled hypertonic saline. Clin Exp Allergy 1994;24(10): Popov TA, Pizzichini MM, Pizzichini E, Kolendowicz R, Punthakee Z, Dolovich J, et al. Some technical factors influencing the induction of sputum for cell analysis. Eur Respir J 1995;8(4): Tzanakis N, Chrysofakis G, Kyriakou D, Bouros D, Siafakas N. Decreased CD4/CD8 T lymphocytes in induced sputum of COPD patients correlates with severity of obstruction (abstract). Eur Respir J 2001;18(suppl. 33):253s.

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