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* From Thoracic Medicine (Drs. Beeh, Culpitt, Giembycz, and Barnes), National Heart and Lung Institute, Faculty of Medicine, Imperial College of Science, Technology & Medicine, London, UK; and Pulmonary Department (Drs. Kornmann and Buhl), Internal Medicine, University Hospital, Mainz, Germany.
Correspondence to: Peter J. Barnes, MD, Thoracic Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College of Science, Technology & Medicine, London SW7 2AZ, UK; e-mail: p.j.barnes{at}ic.ac.uk
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Abstract |
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Design: Sputum supernatant of 20 patients with COPD was used as chemoattractant in a 96-well chemotaxis chamber, with subsequent quantification of migrated cells by a luminescence assay. The contribution of IL-8 and LTB4 to chemotaxis was determined by addition of a neutralizing antibody and a selective receptor antagonist, respectively.
Measurements and results: COPD sputum caused neutrophil chemotaxis in a concentration-dependent manner, with a maximum response evoked with a 10-fold dilution of the original sample. Pretreatment of sputum or neutrophils with either an anti–IL-8 antibody or the LTB4 antagonist, SB 201146, led to a concentration-dependent inhibition of sputum-induced neutrophil chemotaxis, with a maximum suppression (mean ± SEM) of 29.2 ± 4.9% (p < 0.001) from baseline by 100 ng/mL of anti–IL-8 antibody, and 45.6 ± 7% (p < 0.02) by 10 µmol/L of SB 201146. The combination of the anti–IL-8 antibody and SB 201146 inhibited neutrophil chemotaxis, but this was not significantly greater than the effect of SB 201146 or anti–IL-8 alone.
Conclusions: These data confirm the importance of IL-8 and LTB4 as chemoattractants for neutrophils in bronchial secretions from patients with COPD, and suggest that specific inhibitors may have therapeutic potential in COPD.
Key Words: chemotaxis • COPD • human neutrophils • induced sputum • interleukin 8 • leukotriene B4
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Understanding the mechanisms leading to airway neutrophilia in COPD is important, since several chemoattractants may serve as targets for future anti-inflammatory drugs. These anti-inflammatory agents may potentially reduce the rate of decline of lung function in these patients by ameliorating neutrophil-mediated tissue destruction. Several neutrophil chemotactic factors have been implicated, including interleukin (IL)-8, tumor necrosis factor-
,8
and leukotriene B4 (LTB4), with a predominant role for IL-8 in cystic fibrosis,9
LTB4 in bacterial exacerbations of COPD,10
and a combination of both IL-8 and LTB4 in bronchiectasis.7
Moreover, unlike IL-8,11
LTB4 has also been shown to prolong neutrophil survival by inhibiting apoptosis.12
To further investigate the contribution of IL-8 and LTB4 to neutrophil chemotaxis in stable COPD, we used sputum supernatant from steroid-naive COPD patients as a chemoattractant. The roles of LTB4 and IL-8 were determined using a selective LTB4 receptor antagonist, SB 201146,13
and a monoclonal anti–IL-8 antibody, respectively.
There is still ongoing debate about the benefit of inhaled or oral corticosteroids in patients with stable COPD.14 While corticosteroids have no effect on neutrophilic inflammation,15 16 they may influence cytokine levels,17 18 or neutrophil survival and activation.19 20 21 Hence, only steroid-naive patients were chosen to exclude a possible influence of concomitant anti-inflammatory treatment on sputum chemotaxis.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sputum Induction
Sputum induction was performed according to a method previously described.23
Briefly, patients received two puffs of salbutamol (100 µg/puff) 15 min prior to the procedure, and then inhaled 4% hypertonic saline solution delivered by an ultrasonic nebulizer device (Ultra Neb 2000; DeVilbiss; Wolleston, UK) for 15 min. Patients were told to rinse their mouth, blow their nose, and carefully cough sputum into a Petri dish using forced expiratory maneuvers. The first portion of sputum was discarded, and the inhalation procedure was continued for a further 15 min. Lung function was carefully monitored by spirometry every 5 min during induction to ascertain safety of the procedure. Induction was stopped when the total length of induction of 15 min was completed or a > 15% drop in FEV1 occurred. In the latter scenario, patients were administered two additional puffs of salbutamol.
Sputum Processing and Counting
Sputum plugs were closely examined by light microscopy to ascertain least possible contamination of sputum with squamous cells. An appropriate sample was then filled into a 1-mL Eppendorf tube, weighed, and mixed with an equal volume of 0.1% dithiothreitol (Calbiochem; Schwalbach, Germany) in phosphate-buffered saline solution (PBS) [Gibco Life; Paisley, Scotland]. Sputum was gently vortex mixed and placed into a water bath at 37°C for 15 min to allow homogenization of the sample. This procedure diluted the sputum and concentration of dithiothreitol twofold. Samples were centrifuged (2,800 revolutions per minute for 10 min), the supernatant aspirated and recentrifuged (3,000 revolutions per minute for 5 min) to completely remove cellular components, and immediately frozen at - 70°C. Sputum cells were counted after cytospin preparation and staining with Hemacolor staining (Merck; Darmstadt, Germany) to assess the quality of the sample. Only supernatant of sputum samples with a squamous cell contamination of < 20% was used for further analysis.
Quantification of Chemoattractants in Sputum Supernatant
The concentration of IL-8 and LTB4 pooled sputum supernatant was measured by commercially available immunoassays (IL-8, R&D Systems; Abingdon, UK, and LTB4, Amersham Pharmacia; Amersham, UK). Due to insufficient sample quantities, it was not possible to determine concentrations in each individual patient. Hence, data represent concentrations in the sputum pool that was consecutively used for chemotaxis experiments (10-fold dilution).
Isolation of Blood Neutrophils
Neutrophils were harvested from peripheral blood samples of healthy, nonsmoking, nonatopic donors using a discontinuous Percoll gradient (centrifugation at 400g for 25 min at 18°C). Neutrophils were harvested from the 70/80% interface, washed twice with Hanks’ balanced salt solution (HBSS) [Sigma Aldrich Chemicals; Poole, UK], containing 20 mM of hydroxylethyl piperazine-ethanesulfonic acid (HEPES) [Sigma Aldrich Chemicals] and 0.1% bovine serum antigen (BSA) [Sigma Aldrich Chemicals], and resuspended in HBSS 20 mM of HEPES and 0.1% BSA, at a cell concentration of 106/mL. Neutrophils were consistently > 95% pure, assessed by counting of May-Gruenwald-Giemsa–stained cytospins, and > 95% viable, assessed by trypan blue staining.
Neutrophil Chemotaxis Assay
Neutrophil chemotaxis was assayed using a 96-well microchemotaxis chamber (Neuro Probe; Gaithersburg, MD).24
Briefly, a total of 100,000 neutrophils were pipetted into each upper well of the chemotaxis chamber, with a 3-µm pore diameter filter membrane (Neuro Probe) sealed underneath. Medium containing sputum supernatant, defined chemoattractants, or cell standards in serial dilutions were placed in a 96-well cell culture plate (Packard; Pangbourne, UK) under the filter, ensuring contact between medium and filter. Migrating cells were collected in the cell culture plate and centrifuged (1,700 revolutions per min for 15 min). The supernatant was removed, and 50 µL of mammalian cell lysis solution (ATPLite; Packard) was added for 15 min followed by 50 µL of substrate solution (ATPLite). Luminescence was read 10 min later using a Lucy II microplate reader (Anthos; Salzburg, Austria). Standard curves were generated according to the luminescence of the serial cell dilutions from which the number of migrated cells per well was interpolated.
Validation of the Chemotaxis Assay and Activity of Inhibitors
The chemotaxis assay was validated by determining whether neutrophils migrated to known chemoattractants. Neutrophil chemotaxis to IL-8 (rhIL-8; R&D Systems) and LTB4 (Sigma Aldrich Chemicals) diluted serially was concentration dependent, reliable, and reproducible.
To confirm the capacity to specifically inhibit chemoattractants used in the assay, we have used a LTB4 receptor antagonist (SB 201146; SmithKline Beecham; King of Prussia, PA), and a commercially available monoclonal anti–IL-8 antibody neutralizing the biological activity of IL-8 (R&D Systems) in a series of inhibition experiments. SB 201146 is a first-generation trisubstituted LTB4 receptor antagonist with high affinity and no demonstrable agonist activity up to a concentration of 10 µmol/L in vitro. The anti–IL-8 antibody is a mouse IgG1 anti-human IL-8 antibody suitable for bioactivity neutralization assays (according to the brochure of the manufacturer).
Inhibitory capacity was confirmed in neutrophil chemotaxis assays using optimal concentrations of LTB4 and IL-8 as chemoattractants. Thus, IL-8 (10 µg/mL)-induced migration was assessed after 1 h at 37°C in the absence and presence of the anti–IL-8 antibody (10 pg/mL to 10 µg/mL) in HBSS containing 20 mM HEPES and 0.1% BSA. Similarly, chemotaxis effected by LTB4 (100 nm) was determined under identical experimental conditions in the absence and presence of SB 201146 (1 nmol to 10 µmol/L).
Assessment of Optimal Sputum Concentration for Chemotaxis Experiments
The optimal concentration of sputum supernatant that was consecutively used in further chemotaxis assays was determined empirically by performing serial dilutions of (1:5, 1:10, 1:20, 1:40, 1:80, and 1:160) of the original sample.
Determination of the Contribution of IL-8 and LTB4 to Sputum-Induced Chemotaxis
Serial dilution (10 pg/mL to 1 µg/mL) of the anti–IL-8 antibody or SB 201146 (10 nmol to 100 µmol/L) were added to either sputum (anti–IL-8) or polymorphonuclear leukocyte suspension (SB 201146) at the optimal dilution for 1 h at 37°C before neutrophil chemotaxis was measured. In further experiments, the maximally effective concentrations of SB 201146 and anti–IL-8 antibody were used in combination.
Statistical Analysis
Statistical analysis was performed using the STATA 5.0 software package (Stata Corporation; College Station, TX). Data are presented as the mean ± SEM of five independent experiments. The effect of the anti–IL-8 antibody and SB 201146 on neutrophil chemotaxis was evaluated by one-way analysis of variance using the Bonferroni multiple comparison tests. All p values < 0.05 were considered significant.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-8 and LTB4-Induced Neutrophil Chemotaxis
Under resting conditions or after exposure to diluent, human neutrophils underwent spontaneous chemotaxis affecting 0.4 ± 0.2% of the total cell population after 1 h. The addition of IL-8 and LTB4 augmented chemotaxis over the spontaneous response in a concentration-dependent manner with the effective concentration causing a 50% fall (EC50) values of 4 ng/mL and 3.3 nmol, respectively (Fig 1
). The concentration-response curves that described IL-8–induced directional migration were shallow, with a maximal effect elicited between 10 ng/mL and 100 ng/mL (Fig 1 , left, a). In addition, neutrophil responsiveness to IL-8 varied between donors as evinced from the large SEs (Fig 1
, left, a). In contrast, LTB4-induced chemotaxis was described by typical and reproducible sigmoidal concentration-response curves with a maximal effect seen at 100 nmol. At the maximally effective concentration of IL-8 and LTB4, the number of neutrophils that migrated as a percentage of the total number of cells was approximately 20 to 25% (Fig 1)
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Neutrophil Chemotaxis Evoked by Sputum From COPD Patients
Sputum was induced in patients with established COPD, and its ability to promote neutrophil chemotaxis was determined. The relationship that described sputum-induced chemotaxis was bell shaped, with a maximal response seen with sputum diluted 1:10 in PBS (Fig 3
). This concentration of sputum, which was used in experiments to determine the contribution of IL-8 and LTB4 to the chemotactic activity, promoted a chemotactic response (22,586 ± 5,807 cells per well) that was comparable in magnitude to that effected by 100 ng/mL IL-8 (21,303 ± 6,871 cells per well) and 100 nmol LTB4 (23,970 ± 6,159 cells per well). As shown in Figure 3
, sputum more or less concentrated was significantly less able to promote directional neutrophil migration.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The methodology was validated by studying the effect of defined concentrations of chemoattractants and blocking these responses with a receptor antagonist (for LTB4) and a neutralizing monoclonal antibody (for IL-8). Neutrophils from healthy donors migrated in a concentration-dependent fashion in response to both IL-8 and LTB4, and this effect was reduced by both the anti–IL-8 antibody and SB 201146. It was reasoned that these selective inhibitors should also suppress neutrophil chemotaxis evoked by sputum induced from patients with COPD, since both IL-8 and LTB4 have been detected in bronchial secretions from these patients.8 25 Moreover, there is a correlation between these chemoattractants and the degree of airway or sputum neutrophilia in asthma and COPD.2 8 26 27
In this study, we show that IL-8 and LTB4 are present in sputum supernatant from patients with stable COPD. Moreover, sputum from COPD patients is, indeed, chemoattractant for peripheral blood neutrophils and that optimal chemotactic activity was seen when sputum was diluted 1:10 in PBS. Sputum more or less concentrated was markedly less effective at promoting directional neutrophil migration. These observation concur with a recent study reported by Mikami et al,7 who investigated sputum-induced chemotaxis of neutrophils from patients with bronchiectasis. In that study, the maximum chemotaxis was elicited when sputum was diluted sixfold to eightfold. The reason(s) why more concentrated sputum samples were less chemotactic is unclear, but our data are consistent with previous findings where agonist-induced neutrophil chemotaxis was described by bell-shaped concentration-response curves28 29 possibly indicative of receptor desensitization. Indeed, in the present study, there was a reduced tendency of LTB4 to evoke chemotaxis at high concentrations. However, it is unlikely that this explanation fully accounts for the effect, as it was not observed with IL-8 at a concentration (100 ng/mL) that is rarely found in the sputum of patients with COPD,9 15 26 and also exceeded the IL-8 levels of pooled sputum in our study (20.9 ng/mL). It is possible that LTB4 and/or other chemoattractants present in sputum could be inhibitory at high concentrations. Alternatively, low concentrations of an endogenous inhibitor(s) could produce the same effect.
Unfortunately, the aforementioned data are limited by the fact, that we were only able to quantify IL-8 and LTB4 in pooled sputum, the reason for this being the large amount of sputum supernatant required for the chemotaxis assay. Therefore, it was practically impossible to sample sufficient quantities of supernatant to perform measurements of mediators on an individual basis. However, this limitation does not affect the basic findings of our study, since it was focused on the functional activity, not the mere presence of these mediators in sputum, and the data of pooled sputum clearly confirm the presence of IL-8 and LTB4 in the samples used.
In our study, dithiothreitol was used for sputum processing. Dithiothreitol has been shown to alter the expression of cell surface markers,30 but data on the influence of dithiothreitol on mediator recovery from sputum are inconclusive.31 32 To our best knowledge, an impact of sputum processing with dithiothreitol on chemotactic activity has not been demonstrated. Despite the fact that we have not systematically analyzed a possible effect of dithiothreitol on chemotaxis in our study, a considerable effect on the results is very unlikely, since all sputum samples were treated equally, and inhibition experiments were performed with the same samples.
Studies with anti–IL-8 antibody and SB 201146 confirmed that IL-8 and LTB4 are important factors contributing to the chemotactic activity of COPD sputum, respectively. Indeed, these series of studies indicated that IL-8 and LTB4 accounted for 29% and 45% of the chemotactic activity, respectively. The inhibitory effect of anti–IL-8 is comparable to that reported by Mikami et al,7 but significantly lower than that found by Richman-Eisenstat and colleagues.9 The reason for this discrepancy remains unclear. However, we validated our methodology in a manner similar to that described by Mikami et al,7 and thus the differences may be methodologic. In contrast, the suppression of sputum-induced neutrophil chemotaxis by SB 201146 was more marked than that reported by other investigators.7
Pretreatment of neutrophils with a combination of SB 201146 and anti–IL-8 attenuated sputum- induced neutrophil chemotaxis by approximately 45%, which was less than the combined effect of either drug alone. These data may suggest that LTB4 is of greater relevance for the recruitment of neutrophils in COPD than has been reported in other chronic lung diseases including cystic fibrosis and bronchiectasis, and demonstrate that COPD sputum contains other, as yet unidentified, neutrophil chemoattractants.33
In conclusion, this study confirms that IL-8 and LTB4 contribute to neutrophil chemotaxis effected by sputum obtained from patients with established, stable COPD. The data also clearly show that as yet unidentified chemoattractants are also involved in neutrophil migration. These findings highlight the potential of novel therapeutic approaches aimed at suppressing chronic neutrophilic inflammation in patients with COPD. However, whether these approaches will prove to be efficient and safe in humans remains questionable. The pivotal importance of IL-8 in eliciting a host-defense response to microbiological infections raises a critical issue regarding the safety and tolerability of a therapeutic approach aimed at blocking the effect of IL-8 in patients with COPD. Thus far, humanized monoclonal anti–IL-8 has been shown to be well tolerated in preclinical studies, but data in humans have not been reported.34 Since this issue relates to virtually all neutrophil chemoattractants, inhibition of LTB4 by specific antagonists also needs to be carefully scrutinized with regards to safety and tolerability, before it can be considered a potential anti-inflammatory treatment in patients with COPD.
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Footnotes |
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Received for publication November 15, 2001. Accepted for publication July 17, 2002.
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References |
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and IL-8, in induced sputum of non-asthmatic patients with dry cough. Thorax 1999;54,234-237 induces additive neutrophil migration. Allergy Asthma Proc 1999;20,361-363[CrossRef][ISI][Medline]
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