Impaired Neutrophil Chemotaxis in Chronic Obstructive Pulmonary Disease (COPD)

Transcription

1 Impaired Neutrophil Chemotaxis in Chronic Obstructive Pulmonary Disease (COPD) Takahiro Yoshikawa, Gordon Dent, Jon Ward, Gilbert Angco, Guangmin Nong, Naho Nomura, Kazuto Hirata, and Ratko Djukanovic Online Data Supplement

2 METHODS Subjects Three groups of volunteers were studied in two series of experiments: 28 subjects with COPD, 16 age-matched healthy smokers and 17 age-matched healthy non-smokers. Volunteers with COPD were further categorized according to GOLD criteria as stage I (n=9), with FEV 1 80% predicted and FEV 1 /FVC<70%, and COPD stage II IV (n=19), using the following criteria: stage II 50% FEV 1 <80% predicted and FEV 1 /FVC<70%; stage III 30% FEV 1 <50% predicted and FEV 1 /FVC<70%; stage IV FEV 1 <30% predicted and FEV 1 /FVC<70% (1, 2). None of the subjects had respiratory failure. Volunteers with bronchiectasis and other airway diseases, as well as those with positive skin prick tests to any of the eight common aeroallergens (Aspergillus fumigatus, mixed grass pollens, mixed tree pollen, house dust mite [Dermatophagoides pteronyssinus], Dermatophagoides farinae, dog hair and cat fur) were excluded to avoid confounding effects of chronic infection and atopy, respectively. Subjects who had used inhaled or oral corticosteroids or had had a respiratory infection or an exacerbation within 4 weeks prior to the study were also excluded. Three subjects from the COPD stage II-IV group were treated with bronchodilators (two with salbutamol as required and one with ipratropium bromide). No other medications were taken within 4 weeks preceding the E1

3 study. Forced expiratory volume in one second (FEV 1 ) was measured according to the ATS guidelines (2). Bronchodilator responsiveness to 400 µg of salbutamol was assessed and post-bronchodilator spirometric values were recorded. All healthy non-smokers were lifelong non-smoking volunteers who had no history of lung disease. All healthy smokers and COPD subjects in the present study had a history of current smoking. COPD subjects all had a clinical history of respiratory symptoms, such as chronic bronchitis (defined as productive cough on most days for three months of the year for at least 2 years), and exertional dyspnea depending on disease severity. In addition, these subjects had an irreversible airflow limitation (FEV 1 /FVC<70% and bronchial reversibility <12% predicted FEV 1 after inhalation of bronchodilator). Sputum induction and processing Sputum was induced and processed as recommended by the European Respiratory Society s Task Force on induced sputum (3) using 4.5% hypertonic saline delivered via an ultrasonic nebulizer (Ultra-Neb 2000; DeVilbiss Corp., Somerset, PA) for a total of 20 min. Expectorated samples were processed immediately. In order to reduce salivary contamination, mucoid portions of the sputum samples originating from the lower respiratory tract were carefully selected and weighed. A small quantity of sputum was set E2

4 aside for processing with dithioerythritol (DTE; 1 volume of 10 mm DTE per volume of sputum) for differential cell counts. The rest of the selected sample was processed with phosphate-buffered saline (PBS; Oxoid Ltd., Basingstoke, UK) and used for chemotaxis experiments. PBS rather than DTE was used because of concern that processing with DTE might reduce the disulfide bonds of chemokines and thus alter their function (4). These samples were diluted 1:4 in sterile PBS and a cocktail of protease inhibitors (Sigma- Aldrich Chemical Co., Poole, UK; 22.5 µl of protease inhibitors per mg of sputum) was added. All processed samples were stored at -80 C until analysis. Prior to chemotaxis experiments, the supernatants were subjected to sonication and high-speed centrifugation (12,000 g for 2 min) to further clarify the samples and thus improve sample homogeneity. Isolation and fluorescent labelling of peripheral blood neutrophils Neutrophils for the first series of experiments were isolated from blood using established methods (5). Erythrocytes were sedimented with dextran (mean MW 180,000; 6% solution in PBS) at room temperature for 45 min. The leukocyte-rich plasma was layered over a density gradient (Lymphoprep g/ml, AXIS-Shield, Oslo, Norway) and centrifuged at 800 g for 25 min. The granulocyte-rich pellet was removed and residual erythrocytes lysed with ice-cold sterile water. The chemotactic responses of neutrophils separated in this way were compared with E3

5 the responses obtained using the purification method of Haslett and colleagues (6). Briefly, peripheral blood (40 ml aliquots) was collected and anti-coagulated with sodium citrate (3.8% w/v), then centrifuged at 270 g for 20 min at 22 o C. The platelet-rich plasma supernatant was collected and centrifuged at 2,100 g for 20 min to provide platelet-poor plasma (PPP), and the remaining blood cell fraction was mixed with a 6% dextran-500 solution (w/v, 1:5 ratio), then allowed to settle to sediment erythrocytes under gravity (40 min at 22 o C). The leukocyte-rich upper layer was carefully aspirated and centrifuged at 270 g for 7 min. The resulting leukocyte-rich pellet was resuspended in PPP then underlayered with 42% (v/v) and 51% (v/v) discontinuous PPP-Percoll gradients (freshly prepared from 90% (v/v) Percoll in 0.9% saline and PPP. Neutrophils were separated from other cell types by centrifugation of the gradients at 170 g for 12 min at 18 o C. After centrifugation, the mononuclear cell layer was discarded and the neutrophils found at the 42% 51% PPP-Percoll interface were harvested then sequentially washed with 50% PPP- PBS solution, followed by Dulbecco s phosphate-buffered saline (PBS). The neutrophils (>95% pure, >98% viability, as determined by exclusion of trypan blue) were resuspended in Hanks balanced salt solution (HBSS) (Invitrogen, Paisley, UK) containing 10 mm HEPES (Invitrogen) and 0.35% bovine serum albumin (Sigma) ( chemotaxis buffer ). The cells were then fluorescently labelled with calcein (Molecular E4

6 Probes, Portland, OR) as described (7, 8). The cell suspension was washed twice with chemotaxis buffer, adjusted to cells/ml in chemotaxis buffer, and further incubated at 37 C, 5% CO 2 for 45 min, after which they were ready for chemotaxis experiments. Neutrophil chemotaxis Neutrophil chemotaxis was quantified in a fluorescence-based assay using a 96-well microplate-based chemotaxis chamber (Neuroprobe, Gaithersburg, MD) and 3-µm poresize polyvinylpyrrolidone (PVP)-coated polycarbonate filters (9, 10). The chemotactic properties of peripheral blood neutrophils from all the subject groups were first assessed using fmlp and IL-8. Custom microplate wells (30 µl well volume) were loaded with chemotaxis buffer (negative control for chemoattractants), chemoattractant (fmlp M, or IL M), PBS (negative control for sputum), or sputum fluid phase at the indicated dilutions. A self-adhesive 3-µm pore-size PVP-coated polycarbonate filter was fixed on top of the plate, which was then preincubated for 15 min at 37 C, 5% CO 2. A range of cell standards ( ) were added to one column of wells to allow quantification of migrated cells at the end of the assay. Calcein-labeled neutrophils (250,000 cells in 50 µl) were placed into the upper wells of the chemotaxis chamber and the chamber was incubated at 37 C, 5% CO 2 for 60 min. E5

7 After incubation, the neutrophil suspension in each upper well was replaced with cell detachment buffer (cold PBS containing 2 mm EDTA and 0.5% BSA) and the chamber was incubated at 4 C for 30 min to detach neutrophils from the lower surface of the filter (11). The microplate was then removed and centrifuged at 200 g for 10 min before removing the membrane filter. Тhe filter was removed and the fluorescence in the wells was measured in a fluorescence plate-reader (FLx800, Bio-Tek Instruments Inc., Winooski, VT; λ ex =485 nm, λ em =530 nm). Numbers of migrating cells were calculated from the fluorescence measurements by linear interpolation using the standard curve. The directional chemotaxis to sputum was assessed as described above for IL-8 and fmlp using sputum samples serially diluted in PBS at 1:10, 1:20, 1:40 and 1:80. This was also done in two series of experiments: in the first series, neutrophils from one healthy non-smoker were used in order to provide a constant for the responder cell element of the experiment, allowing differences in chemotactic responses that are solely due to differences in sputum donors to be assessed. In order to validate the results of this first series on neutrophils from more than one donor, another series of experiments was performed using 9 healthy non-smokers as donors of neutrophils and assessing the response of these to the same sputum samples used in series 1. In all cases fmlp was used as control and in all experiments this response was as expected (data not shown) E6

8 RESULTS Correlation of inflammatory indices and lung function Sputum neutrophil numbers showed a significant negative correlation with FEV 1 across all subject groups, indicating an association between airway neutrophilia and impaired lung function (Figure E1). In contrast, fmlp-induced migration of blood neutrophils showed significant positive correlation with FEV 1 (Figure E2). The reproducibility of the chemotactic responses to these two chemoattractants was tested on three occasions using two donors. The results, showing high reproducibility, are shown in Figure E3. In order to validate the chemotaxis responses observed when using neutrophils purified with dextran, Lymphoprep and hypotonic lysis, chemotactic responses to the full range of fmlp and IL-8 concentrations were compared using neutrophils obtained by the two methods. The experiments, conducted using two donors showed remarkable similarity across the whole range of concentrations (Figure E4). Chemotaxis of blood neutrophils induced by the sputum fluid phase Dilution-response curves for sputum supernatants from healthy subjects (non-smokers and smokers) were not bell-shaped, but showed a dilution-dependent response. In contrast, E7

9 sputum supernatants from COPD subjects did exhibit shallow, bell-shaped dilutionresponse curves (Figure E5). Bland-Altman analysis was performed to test the validity of comparing chemotaxis data obtained in the two series of experiments. This analysis allows determination of systematic biases between two methods applied to the same measurement (12). Comparison of sputum chemotactic activity determined using blood neutrophils obtained by different protocols in the two series of experiments showed no significant bias toward greater responsiveness in one series, with only 2/87 determinations (2.3%) lying outside the limits of the coefficient of repeatability, and no trend in difference with the magnitude of the response (Figure E6). In common with spontaneous and fmlp-induced neutrophil migration, peripheral blood neutrophil migration in response to sputum fluid phase from all subjects exhibited a significant positive correlation with FEV 1 (Figure E7). Thus, while impaired lung function in COPD is associated with increased sputum neutrophil numbers, it is also associated with both reduced sputum chemotactic activity for neutrophils and reduced migratory activity of peripheral blood neutrophils. These results support our finding that neutrophil chemotactic responsiveness and sputum chemotactic activity are reduced in subjects with COPD. E8

10 Online Figure Legends E1 Correlation between sputum neutrophil counts (as percent of total inflammatory cells) and FEV1 (% predicted). E2 Correlation between fmlp (10-8 M)-induced neutrophil migration and FEV1 (% predicted). E3 The reproducibility of the chemotactic responses to fmlp (top) and IL-8 (bottom) tested on three occasions using two neutrophil donors (donor-1 left panel; donor-2 right panel). E4 Comparison of chemotactic responses to the full range of fmlp (left) and IL-8 concentrations (right) using neutrophils purified by two different methods: 1 Percoll, without hypotonic lysis; 2 dextran, Lymphoprep and hypotonic lysis. E5 Neutrophil migration induced in neutrophils from 2 healthy donors by 4 dilutions of sputum samples. Numbers of cells migrating to sputum are shown as median values for E9

11 each group after subtracting the numbers of spontaneously migrating cells: healthy nonsmokers (open circles), healthy smokers (open triangles), subjects with COPD stage I (closed circles) and stage II IV (closed triangles). E6 Bland-Altman plot comparing results from neutrophil chemotactic responses to sputum supernatants in experiments from series 1 and series 2 in which neutrophils from a single and several donors were used, respectively. Data are from 87 determinations using 29 separate sputum samples assayed at 3 dilutions: 1:10 (closed circles), 1:20 (square) and 1:40 ( ). Broken lines indicate mean ±95% limits of agreement (1.96 standard deviation.) E7 Correlation between sputum chemotactic activity (expressed as migration of healthy nonsmoker neutrophils in response to sputum fluid phase) and FEV1 (% predicted). E10

12 REFERENCES E1. Pauwels RA, Buist AS, Calverley PMA, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: NHLBI/WHO global initiative for chronic obstructive lung disease (GOLD) workshop summary. Am J Respir Crit Care Med 2001;163: E2. National Heart, Lung, and Blood Institute. Global Initiative for Chronic Obstructive Lung Disease: global strategy for diagnosis, management, and prevention of chronic obstructive pulmonary disease (2004 update). National Institutes of Health, Bethesda E3. Paggiaro PL, Chanez P, Holz O, Ind PW, Djukanovic R, Maestrelli P, Sterk PJ. Sputum induction. Eur Respir J 2002;Suppl 37:3s-8s. E4. Kelly MM, Keatings V, Leigh R, Peterson C, Shute J, Venge P, Djukanovic R. Analysis of fluid-phase mediators. Eur Respir J 2002;Suppl 37:24s-39s. E5. Böyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest 1968;Suppl 97: E11

13 E6. Haslett C, Guthrie LA, Kopaniak MM, Johnson RB, Jr., Henson PM. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol 1985;119: E7. Taylor ML, Dastych J, Sehgal D, Sundstrom M, Nilsson G, Akin C, Mage RG, Metcalfe DD. The Kit-activating mutation D816V enhances stem cell factordependent chemotaxis. Blood 2001;98: E8. Dent G, Hadjicharalambous C, Yoshikawa T, Handy RLC, Powell J, Anderson IK, Louis R, Davies DE, Djukanovic R. Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum. Am J Respir Crit Care Med 2004; 169: E9. Frevert CW, Wong VA, Goodman RB, Goodwin R, Martin TR. Rapid, fluorescencebased measurement of neutrophil migration in vitro. J Immunol Methods 1998;213: E10. Mishra RK, Scaife JE, Harb Z, Gray BC, Djukanovic R, Dent G. Differential dependence of eosinophil chemotactic responses on phosphoinositide 3-kinase (PI3K). Allergy 2005;60: E11. Shi Y, Kornovski BS, Savani R, Turley EA. A rapid, multiwell colorimetric assay for E12

14 chemotaxis. J Immunol Methods 1993;164:154. E12. Bland JM, Altman DG. Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 1995;346: E13

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20 Figure E6 150,000 Series 2 - Series 1 100,000 50, , , , , , , ,000 Mean of series 1 and series 2 E19

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