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COPD exacerbations, inflammation and treatmentBathoorn, Derk

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RIJKSUNIVERSITEIT GRONINGEN

COPD exacerbations, inflammation and treatment

Proefschrift

ter verkrijging van het doctoraat in deMedische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magnificus, dr. F. Zwarts,in het openbaar te verdedigen op

woensdag 21 november 2007om 14.45 uur

door

Derk Bathoorngeboren op 31 januari 1977

te Roden

Promotores:Prof dr H.A.M. KerstjensProf dr D.S. PostmaProf dr G.H. Koëter

Beoordelingscommisie:Prof W. MacNeeProf dr J.W.J LammersProf dr. A.J.M. van Oosterhout

Paranimfen:Drs P.C. BaselmansDrs M.D. Mensing

Bathoorn, ECOPD Exacerbations: inflammation and treatmentThesis University Medical Center Groningen

Printed at Gildeprint

The clinical trials described in this thesis were financially supported byAstraZeneca, the Netherlands and Stichting Astma Bestrijding

Printing and distribution of this thesis financially supported by:AstraZeneca, the Netherlands, Nycomed, Boehringer Ingelheim, GlaxoWellcome, Novartis Pharma B.V., Meda Pharma, Bayer, HealthcarePharmaceuticals, GUIDE, Rijksuniversiteit Groningen, Stichting AstmaBestrijding, Teva Pharma NL, and Janssen-Cilag.

Copyright E. Bathoorn

ISBN: 978-90-367-3134-8

Table of Contents

Chapter

1. General introduction and outline of thesis 13

2. Review: Inflammation and its modifying therapies in COPD exacerbations 21

3. Safety of sputum induction during exacerbations of chronic obstructive pulmonary disease 45

4. Effects of steroid withdrawal on parameters of inflammation 61

5. Change in inflammation during COPD exacerbations 75

6. Anti-inflammatory effects of combined budesonide/formoterol in COPD exacerbations 93

7. Anti-inflammatory effects of inhaled carbon monoxide in patients with COPD: a pilot study 111

8. Summary 127

9. Discussion 133

Nederlandse samenvatting 147

Dankwoord 157

5

General introduction and outline of thesis

7

Introduction

COPD

In the 2005 update of the Global initiative for chronic Obstructive Lung Disease(GOLD) guidelines, chronic obstructive pulmonary disease (COPD) is definedas: ‘a disease state characterized by limitations in lung airflow that are not fullyreversible. The airflow limitation is usually both progressive and associated withan abnormal inflammatory response of the lungs to toxic particles or gases’(1).In the western world, COPD is most commonly caused by exposure to tobaccosmoke (2), but occupational dusts and air pollution also may cause COPD (3-5).In developmental countries indoor pollution by cooking on biomass fuels is animportant additional cause (6). The patients’ first symptoms of COPD aredyspnoea on exertion, coughing, and increased sputum production. COPD is amajor health problem worldwide: the prevalence of COPD in adults above 40years of age is approximately 9-10% (7). COPD is currently the fourth leadingcause of death in the world (8), and its mortality is still rising (9). COPDexacerbations have an important impact on morbidity and mortality. Thein-hospital mortality for patients with an COPD exacerbation is 8-11% and themortality in the first year following hospitalisation is 23-43% (10;11).

Maintenance treatment with inhaled steroids

The mainstay of treatment of COPD are bronchodilators (1). Several trials inpatients with GOLD stage II-IV have investigated the long-term effects ofmaintenance treatment with inhaled corticosteroids on lung function, quality oflife, and exacerbation rates. Inhaled steroids lead to a 30% reduction ofexacerbation rates (12) and improvement of symptoms. They have only modesteffects on lung function: at best, treatment with high dose inhaled corticosteroidsreduces the annual decline in forced expiratory flow in the first second (FEV1)with 9.9 ml, which by itself is generally deemed not to be clinically relevant(12;13). Therefore, inhaled steroids are currently indicated only in patients withmore severe airway obstruction and frequent exacerbations (14).In a recent meta-analysis of inhaled corticosteroids in stable COPD,anti-inflammatory effects were documented as observed by a reduction in totalnumber of airway cells, neutrophils, lymphocytes, and a trend in eosinophils(15). Although it is generally perceived that the beneficial effects of inhaledcorticosteroids should be due to their anti-inflammatory effects, a directrelationship between reduction in airway inflammation and clinical improvementhas not been reported so far.Treatment with the combination of inhaled steroids and long-actingbronchodilators improves lung function decline, symptoms and health relatedquality of life compared with placebo treatment, but reports on its effectscompared with its mono-components alone are conflicting (16;17). Thecombination therapy reduces sputum neutrophil%, sputum eosinophil counts,and biopsy CD8 lymphocytes in stable COPD (18).In summary, the beneficial effects of inhaled steroids in the treatment of COPDare perhaps modest, and certainly not as large in asthma. Despite the modest

8


beneficial effects, withdrawal of inhaled steroids leads in many patients to adeterioration of symptoms, and often to an exacerbation (19-21). When oneconsiders stopping inhaled steroid treatment of a patient with COPD, this shouldbe monitored carefully.

COPD exacerbations

During COPD exacerbations, a sudden deterioration of COPD symptoms for 24hours or more occurs, i.e. increased dyspnoea, productive cough with anincreased volume and/or more purulent sputum, and less specific symptomssuch as malaise, fatigue, and insomnia. There is no consensus about thedefinition of COPD exacerbations though several have been proposed (see table1). Symptoms worsen already a few days before the actual exacerbations starts,and it may take longer than a month before patients have fully recovered, andsome patients do not return completely to their original health status (22).Frequent occurrence of exacerbations is an important feature of COPD:exacerbations of COPD have a large impact on morbidity and mortality, as wellas on the quality of life of the patient (23;24). Approximately 56% of costs forCOPD in the Netherlands are due to the treatment of patients withexacerbations (25). Since additionally the prevalence of COPD is rising, there isan important and growing need from the perspective of patients, doctors, andsociety to develop interventions to optimally prevent and treat exacerbations ofCOPD.

Exacerbations of COPD have different causes: infections of bacterial or viralorigin have been identified as the most important causes of exacerbations:approximately 50-70% of exacerbations are associated with airway infections(26). Air pollution has been described to cause about 10% of exacerbations (5).However, in up to 30 % of exacerbations, the cause remains unknown (27).During COPD exacerbations, the inflammation in the airway is increased,involving increased numbers of eosinophils, neutrophils and lymphocytes(28-30).

It is difficult to obtain information on airway inflammation during COPDexacerbations. An elegant non-invasive method to assess the inflammation inthe airways is sputum induction. Sputum is induced by the inhalation ofnebulised saline in an isotonic or hypertonic concentration. This both facilitatesand standardises the coughing-up procedure of sputum, and the results of thegained samples reflect the level of inflammation in the airways (31-33).However, there is also a downside to this procedure: inhalation of isotonic andcertainly of hypertonic saline causes a bronchoconstrictive response in manypatients with COPD (34-38). Since this decrease in FEV1 is generally transientand more severe adverse effects do not occur, sputum induction is consideredto be safe even in stable mild to severe COPD (35;38). However, whethersputum can also be induced safely during exacerbations of COPD is less clear.

COPD exacerbations are generally treated with systemic corticosteroids andshort-acting bronchodilators, with or without antibiotics. The beneficial effects ofsystemic steroids are evidence based (39;40), however the effects are modest

9


and certainly not as evident as in the treatment of asthma exacerbations.Furthermore, the use of systemic steroids might cause adverse effects likehyperglycaemia, osteoporosis, and mood swings (39;41;42). Treatment ofCOPD exacerbations with inhaled corticosteroids might be an alternative forsystemic treatment avoiding some adverse effects. Inhaled corticosteroids havebeen shown to be capable of reducing airflow limitation during hospitalisationsfor COPD exacerbations (43). Steroids might exert their beneficial effect in thetreatment of COPD exacerbation by decreasing the eosinophils, since in stableCOPD steroids reduce the levels of airway eosinophils (15;18), and sincesputum eosinophilia predicts a better response to a short-term steroid treatmentin a stable phase of COPD (44-46).

Treatment of COPD exacerbations with antibiotics is still a point of discussion;the most recent GOLD guidelines advocate that antibiotics should be given topatients with exacerbations of COPD with three of the following cardinalsymptoms: increased dyspnoea, increased sputum volume, increased sputumpurulence, or two of the cardinal symptoms if increased sputum purulence is oneof the symptoms. Furthermore antibiotics should be given to all patients with asevere exacerbation of COPD that requires invasive mechanical intervention (1).

Carbon monoxide

Based on reports of in vitro and in vivo studies, it has become clear that carbonmonoxide has potent anti-inflammatory and anti-oxidant capacities. Carbonmonoxide is endogenously generated by the degradation of heme, which isinduced by tissue injury or inflammation (47-49). This degradation is catalyzedby the stress inducible enzyme heme oxygenase-1 (HO-1). In vitro studies haveshown that carbon monoxide downregulates pro-inflammatory cytokines byinhibiting the mitogen-activated protein kinase pathway (50;51). In vivo studiesin several animal species consistently show that inhaled carbon monoxide hasa protective effect against ischemic injury, hyperoxic injury, graft versus hostreactions, and pulmonary inflammation (52-60). These capacities might be oftherapeutic use in respiratory inflammatory diseases (48).The next step is toestablish these beneficial effects in humans.The HO-1 expression in alveolar macrophages in smoking COPD patients isdecreased compared to smokers without COPD (61), and the HO-1 expressionin ex-smokers with COPD is decreased compared to healthy ex-smokers (62).This suggests that the HO-1 is insufficiently up regulated in patients withCOPD. In ex-smokers with COPD, there is an ongoing inflammation even aftersmoking cessation. This inflammation is characterized by increased numbers ofeosinophils and neutrophils in the sputum of ex-smokers with COPD comparedto healthy ex-smoking subjects (63-66). It is unknown what causes the ongoinginflammation after smoking cessation. We hypothesize that the decreasedHO-1 expression, resulting in an abnormal inflammatory response to particlesand gases in the air, might contribute to the ongoing inflammation. Correctionfor the HO-1 impairment, by inhalation of CO, might therefore reduce thenumbers of inflammatory cells in the airways.

10


Outline of the thesis

In this thesis, the results of two clinical trials exploring the effects of twoinflammation modifying therapies for COPD are described, one in COPDexacerbations, and one in stable COPD but with possible future bearings forexacerbations.

Chapter 2 is a review of observational studies on the changes in airwayinflammation from a stable phase of COPD to the onset of an exacerbation. Theaim of this review is to give insight in the increases in the different types ofinflammatory cells, causes of the exacerbation, and whether these inflammatorychanges lead to a decline in lung function. With this insight, we review theinflammation modifying therapies which are currently used, and speculate onfuture therapies to treat COPD exacerbations.

The first of the two trials is the Symbexco-study, which stands for SYMbicort inthe treatment of EXacerbations of COPD. The aim of this trial was to comparethe anti-inflammatory effects of combined budesonide and formoterol versusplacebo in the treatment of COPD exacerbations. The design of this trial isshown in figure one.

At the first visit, we included patients with COPD in a stable phase, measuredinflammation in induced sputum and blood, performed lung function tests, andassessed the patients’ health state. After this visit, inhaled corticosteroids werewithdrawn if used. At the second visit, these measurements were repeatedprovided the patients were still in a stable phase of COPD. However, we noticedthat many patients deteriorated after the steroid withdrawal, and several evenexperienced an exacerbation. Chapter 4 describes the changes after inhaledsteroid withdrawal, aiming to elucidate the inflammatory mechanisms whichcause the exacerbation.

After visit 2, we waited per protocol for the patients to report an exacerbation.When they reported an exacerbation, they were asked to come to the hospitalfor a third visit. At this visit, we repeated the measurements, including sputuminduction, and started the randomised-controlled treatment. We were concernedabout performing sputum induction in exacerbated patients, since the nebulisedsaline used during sputum induction can cause increased airflow limitation, andpatients who have a COPD exacerbation already experience increasedshortness of breath. Therefore we induced sputum by a more cautious protocolthan usual in patients who had a more severe airflow limitation. Chapter 3describes the safety of our method to induce sputum in patients with COPDexperiencing an exacerbation.

Chapter 5 describes the changes in inflammation from a stable phase of COPD(visit 2, no inhaled steroids), to the onset of an exacerbation. It aims to studywhich inflammatory changes identify a bacterial cause of the exacerbation. Theidentification by inflammatory markers of a bacterial cause of an exacerbation

11


could be helpful in the clinician’s decision of an early and useful initiation ofantibiotics in the treatment of COPD exacerbations.

At visit 3, patients were randomised for 14 days of one of three treatments:combined budesonide and formoterol, oral prednisolone, or placebo. Chapter 6describes the results of these treatments. It aims to compare theanti-inflammatory effects of combined budesonide and formoterol to placebotreatment. Secondary objectives are to assess the effects of thebudesonide/formoterol combination versus placebo on lung function, symptomsand health status, and to compare the side effects of the combination versusactive control with prednisolone.

The second trial was designed to study the anti-inflammatory effects of inhaledlow dose carbon monoxide in patients with COPD in a stable phase (see figure2). Chapter 7 describes the results of this pilot. This is the first exploration of theeffects in humans and hence the group studied is small. Investigating theanti-inflammatory effects on airway inflammation in stable COPD patients is afirst step towards exploring the therapeutic effects of carbon monoxide.

Table 1: Definitions of a COPD exacerbation._______________________________________________________________________________

Davies et al (67) A history of increased breathlessness and at least two ofthe following symptoms for 24 h or more: increased coughfrequency or severity, increased sputum volume orpurulence, and increased wheeze.

Rodriguez-Roisin(68)

A sustained worsening of the patient's condition, from thestable state and beyond normal day-to-day variations, thatis acute in onset and necessitates a change in regularmedication in a patient with underlying COPD

Anthonisen et al (69) A disease state characterized by an increase in symptomsof dyspnea, sputum volume and sputum purulence.Exacerbation types graded on the basis of combinations ofmajor and minor symptoms.

Madison et al (70) An acute tracheobronchitis, generally infectious in aetiology,that occurs in a patient with established COPD.

Pauwels et al (71) Increased dyspnea, cough, or sputum expectoration (qualityor quantity) that led the subject to seek medical attention

Seemungal et al (72) An exacerbation was diagnosed if the following symptompatterns were experienced for at least two consecutivedays: either two or more of three major symptoms (increasein dyspnea, sputum purulence, and increased sputumvolume); or any one major symptom together with any oneof the following minor symptoms: increase in nasaldischarge, wheeze, sore throat, cough, or fever.

GOLD 2006 (1) An event in the natural course of the disease characterizedby a change in the patient’s baseline dyspnea, cough,and/or sputum that is beyond normal day- to-day variations,is acute in onset, and may warrant a change in regularmedication in a patient with underlying COPD

12


Figure 1: Design Symbexco-study

LF: Lung function testsSI: Sputum induction

Visit 1: Inclusion. After the visit; inhaled steroids are withdrawn if used.Visit 2: Stable phase without steroids. After this visit, long-acting bronchodilators

if used are replaced by short-acting. Patients are instructed to contactthe research center when experiencing an exacerbation as soon aspossible.

Visit 3: Exacerbation. Patients are randomised for double-blind treatment withSymbicort, prednisolone, or placebo for 14 days. All patients receiveantibiotics (doxycycline), and both ipratropium, and terbutaline as rescuemedication.

Visit 4-6: Visits to evaluate the treatment effects. After visit 6, patients re-startusing their long-acting bronchodilators if used before.

Visit 7 and 8: Follow-up visits. Patients are contacted by telephone to evaluatetheir status of COPD. When a new exacerbation occurs, patients aretreated open label, and this is the end of study. If the patients do nothave a next exacerbation within 90 days after the start of theexacerbations, this is the end of study.

13


Figure 2: Design of carbon monoxide in COPD-study.CO: carbon monoxide inhalation for 2 hours per day; Pla: placebo inhalation for 2 hours per day;LF: lung function; PC20: provocative concentration of methacholine causing a 20% fall in FEV1.Lung function, sputum and blood were assessed 17 hours after the last inhalation of CO or placebo.

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(61) Maestrelli P, Paska C, Saetta M, Turato G, Nowicki Y, Monti S et al. Decreased haemoxygenase-1 and increased inducible nitric oxide synthase in the lung of severe COPDpatients. Eur Respir J 2003; 21(6):971-976.

(62) Slebos DJ, Kerstjens HA, Rutgers SR, Kauffman HF, Choi AM, Postma DS. Haemoxygenase-1 expression is diminished in alveolar macrophages of patients with COPD.Eur Respir J 2004; 23(4):652-653.

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(63) Rutgers SR, Postma DS, ten Hacken NH, Kauffman HF, Der Mark TW, Koeter GH et al.Ongoing airway inflammation in patients with COPD who do not currently smoke. Thorax2000; 55(1):12-18.

(64) Turato G, Di Stefano A, Maestrelli P, Mapp CE, Ruggieri MP, Roggeri A et al. Effect ofsmoking cessation on airway inflammation in chronic bronchitis. Am J Respir Crit CareMed 1995; 152(4 Pt 1):1262-1267.

(65) Willemse BW, ten Hacken NH, Rutgers B, Lesman-Leegte IG, Postma DS, Timens W.Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomaticsmokers. Eur Respir J 2005; 26(5):835-845.

(66) Lapperre TS, Postma DS, Gosman MM, Snoeck-Stroband JB, ten Hacken NH, HiemstraPS et al. Relation between duration of smoking cessation and bronchial inflammation inCOPD. Thorax 2006; 61(2):115-121.

(67) Davies L, Angus RM, Calverley PM. Oral corticosteroids in patients admitted to hospitalwith exacerbations of chronic obstructive pulmonary disease: a prospective randomisedcontrolled trial. Lancet 1999; 354(9177):456-460.

(68) Rodriguez-Roisin R. Toward a consensus definition for COPD exacerbations. Chest2000; 117(5 Suppl 2):398S-401S.

(69) Anthonisen NR, Manfreda J, Warren CP, Hershfield ES, Harding GK, Nelson NA.Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease. Ann InternMed 1987; 106(2):196-204.

(70) Madison JM, Irwin RS. Chronic obstructive pulmonary disease. Lancet 1998;352(9126):467-473.

(71) Calverley PM, Anderson JA, Celli B, Ferguson GT, Jenkins C, Jones PW et al.Salmeterol and fluticasone propionate and survival in chronic obstructive pulmonarydisease. N Engl J Med 2007; 356(8):775-789.

(72) Seemungal TA, Donaldson GC, Paul EA, Bestall JC, Jeffries DJ, Wedzicha JA. Effect ofexacerbation on quality of life in patients with chronic obstructive pulmonary disease. AmJ Respir Crit Care Med 1998; 157(5 Pt 1):1418-1422.

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Chapter 2

Airways inflammation and its treatment during acuteexacerbations of COPD

Erik Bathoorn1, Huib Kerstjens1, Dirkje Postma1, Wim Timens2,William MacNee3

1Groningen Research Institute for Asthma and COPD (GRIAC),Department of Pulmonology, and 2Department of Pathology,University Medical Center Groningen, University of Groningen, theNetherlands. 3Edinburgh Lung and the Environment GroupInitiative/Colt Research Laboratories, Medical School, University ofEdinburgh, Edinburgh, Scotland, United Kingdom.

Accepted by Int. Journal of COPD

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Introduction

Exacerbations are an important feature of COPD, since they result indeterioration of a patient’s quality of life (1), contribute to decline in lung function(2), and lead to over 50% of all COPD related costs (3;4). A disease state withsuch important consequences is in need of a tight definition, yet there is noconsensus on such a definition. Several definitions have been proposed,focussing mostly on symptoms, sometimes in combination with infectiousaetiology (5-7). The most quoted definition is the one proposed by Anthoniseni.e. a disease state characterized by an increase in symptoms of dyspnea,sputum volume and sputum purulence (8). Although many physicians consideran increase in inflammation as a core feature of an acute COPD exacerbationand indeed several studies provide evidence that this is the case, (see later inthis review), none of the definitions in use for acute exacerbations capture theterm inflammation. A first effort using inflammatory biomarkers to objectivelyconfirm COPD exacerbations has been published recently. This study showedthat plasma C-reactive protein in combination with one major symptom is usefulto confirm an exacerbation of COPD (9). It is important to focus on inflammation,since it gives insight into the pathological changes causing an exacerbation,thereby possibly providing directions for future therapies which modifyinflammation.

Bronchodilators and corticosteroids are the most commonly used drugs to treatexacerbations of COPD. Corticosteroids elicit a very broad array ofanti-inflammatory actions. The use of systemic corticosteroids as treatment inCOPD exacerbations is evidence based (10). The beneficial clinical effects havebeen extensively investigated, but less is known of the underlyinganti-inflammatory effects of corticosteroids in exacerbations of COPD. Inhumans, only one placebo controlled study (published in abstract form (11)) hasinvestigated the anti-inflammatory mechanisms of steroids in COPDexacerbations, showing a suppressive effect of corticosteroids on sputumeosinophils.

It is difficult but certainly not impossible to study aspects of inflammation evenduring acute exacerbations of COPD. For instance, it has been shown thatsputum can safely be induced also in patients with severe airflow limitation(12;13). Furthermore, techniques such as exhaled breath condensate can beapplied but their repeatability is poor and it is uncertain which compartment(luminal, bronchial wall, alveolar, and parenchyma) the measured biomarkersreflect. More invasive assessment of inflammation in specific lungcompartments by bronchial biopsy, broncho-alveolar lavage and theoreticallyeven transbronchial biopsy is severely restricted during acute exacerbations forobvious safety and ethical reasons. Evaluating autopsies of patients who diedduring COPD exacerbations would be very informative, but, to the best of ourknowledge there are no reports of histopathology of lung tissue of patientsdeceased during COPD exacerbation.

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Airways inflammation and its treatment during acute exacerbations of COPD

The information on inflammation in COPD exacerbations is fragmentary so far,because mainly cross-sectional information has been obtained duringexacerbations without information during the prior stable state and onlyoccasionally information after the resolution of the exacerbation. Suchinformation is necessary, since it will allow dissection of the predisposing factors,types of exacerbations, and patterns of resolution. The majority of information oninflammation during exacerbations in COPD is derived from studies usingsputum inductions. Induced sputum is preferred to spontaneous sputum, sincenot all patients produce sputum spontaneously, and induced sputum containshigher percentage viable cells (12).

Many factors have to be taken into account when assessing inflammation inacute exacerbations of COPD, given the mixed aetiology of these COPDexacerbations. The major causes of exacerbations which have been identifiedare viral and bacterial infection, and air pollution (14;15). In approximately onethird of all exacerbations a cause cannot be identified (16). Certainly the differentknown causes of exacerbation will result in various types of inflammation (17).Furthermore, concomitant use of medication such as inhaled or oralcorticosteroids have to be taken into account, since they affect the type ofinflammation (18).

The aim of the current review is to provide a cell-by-cell overview of theinflammatory processes during COPD exacerbations. We will evaluate cellnumbers, activation, and cytokine production, cellular interactions, damagingeffects of inflammatory mediators to tissue, and the relation to symptoms at theonset of COPD exacerbations. We also speculate on future therapeutic optionsto modify inflammation during COPD exacerbations.

Inflammation

Neutrophils

Neutrophil numbers are slightly, but significantly increased in bronchial glands,submucosa and in subepithelial tissue in bronchial biopsies in stable COPD,compared to healthy persons (smokers or non-smokers), and the numbers ofneutrophils positively related to severity of the airflow limitation (19-21). Thelatter might result from bacterial colonisation, which may be present in sputum inthe case of severe airflow limitation (22-24).

In COPD exacerbations, neutrophils are increased in both the submucosa andsubepithelial tissue compared to the stable disease (17;25-29). The presence ofpotential pathogenic micro-organisms in sputum in exacerbations is associatedwith higher neutrophil numbers (30), as in stable disease. Exacerbations areassociated with increased sputum neutrophil numbers and the change inneutrophil numbers correlate with greater decrease in FEV1 duringexacerbations (17;31) (figure 1).

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Figure 1: An increase in neutrophils concurs with a drop in FEV1 during a COPD exacerbation.Data of 64 patients. Reproduced with permission from reference 17.

At the time of the resolution of exacerbations, a decrease in neutrophil numberis associated with eradication of bacteria from sputum (32). A postulatedunderlying mechanism for this neutrophilia is the interaction of bacteria withToll-like receptors on antigen presenting cells and epithelial cells by bacteria,which induces the release of pro-inflammatory cytokines, as with viral infections(33;34). Although neutrophils show a relation with the presence of bacteria inboth stable COPD and exacerbations, the increase in neutrophils is not limited tobacterial exacerbations: neutrophils have been shown to increase also duringexacerbations associated with viral infections and in those without demonstrablepathogens (17).

The most potent chemoattractants of neutrophils are leukotriene B4,interleukin-8 (IL-8), epithelial-derived neutrophil attractant-78, and tumornecrosis factor-alpha (42;25;39). COPD patients with frequent exacerbationshave higher IL-8 levels in sputum in the stable phase compared to patients withinfrequent exacerbations, illustrating the important role of neutrophilchemoattraction in the pathophysiology of COPD exacerbations (35). Therecruitment of neutrophils is facilitated by increased expression of adhesionmolecules on the surface of circulating neutrophils, which are stress-inducibleand up-regulated during COPD exacerbations (36-38). Leukotrienes are also

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very potent chemoattractants of inflammatory cells during COPD exacerbations.Not only leukotriene B4, but also leukotriene E4 is increased duringexacerbations and related to blood oxygen tension and airway obstruction in thecourse of exacerbations (30;39;40).

One of the main functions of neutrophils is their anti-bacterial role. To killbacteria, neutrophils degranulate, releasing myeloperoxidase, a highly reactiveacideous oxidant (41), which is increased during COPD exacerbations, both insputum and serum (42).

COPD exacerbations caused by bacterial infections resulting in increasedsputum neutrophils, often evoke a systemic inflammatory response:inflammatory markers such as circulating neutrophil numbers, CRP, fibrinogen,and serum IL-6 are increased during exacerbations. (9;43;44). Severalmechanisms have been proposed for the origin of the increased systemicinflammation. These include:1) spill over of inflammatory mediators from thepulmonary compartment; 2) an inflammatory reaction to tissue hypoxia; 3) areaction induced by the pro-inflammatory bacterial product lipopolysaccharide(45).

Systemic inflammation could be important in the follow-up of exacerbations,since COPD patients with frequent exacerbations have a smaller reduction ofsystemic inflammatory markers in the recovery of an exacerbation, andnon-recovery of an exacerbation is related to persistently increased systemicinflammation (46). Furthermore, systemic inflammation during COPDexacerbations may induce cardiovascular co-morbidity, by causing haemostasisand thrombosis. However, a relationship between increased inflammationcaused by infections during exacerbations and the risk for cardiovascular heartdisease has yet not been proven (43;47;48).

In conclusion, neutrophils are predominantly increased in more severeexacerbations caused by bacterial infections, however this increase is not limitedto bacteria-associated exacerbations alone.

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Table 1: Studies reporting increased airway neutrophils during exacerbations compared to stablephase.

Study Number ofpatients instablephase/exacerbation

FEV1 % ofpredicted instable phase/exacerbation

Number of sputumneutrophils instablephase/exacerbation

% sputumneutrophils instable phase/exacerbation

Method

Papi et al (17) 64 / 64 49.5 / 39.4 9.5 / 26.7x106/grama

n.r. Sputuminduction

Tsoumakidouet al (26)

12 / 12 40 / n.r. n.r. 83.5 / 98.0a Sputuminduction

Bathoorn et al(27)

39 / 39 61 / 51 3.2 / 7.1x106/mLa

72.5 / 72.0 Sputuminduction

Mercer et al(28)

19 / 12 n.r./37.6 1.48 / 2.19x106/grama


Fujimoto et al(29)

30 / 30 52.9 / 40.6 4.4 / 24.4x106/grama


Balbi (137) 8 / 5 71 / 64 10 / 83x103/mLa

n.r. / n.r. BAL

_______________________________________________________________________________a: p <0.05n.r.: data not reported

Eosinophils

Eosinophilic inflammation is generally not associated with COPD. In a stablephase, there is little evidence for a role of eosinophils except in a specific COPDphenotype, which shows little emphysema and bronchial wall thickening oncomputed tomography scans and a good response to corticosteroids (49-53).This COPD phenotype has many features of asthma, and it would be of interestto compare in airway tissue histology from patients with this particular COPDphenotype and that of asthma patients. During COPD exacerbations it isrecognised that an “asthma-like” inflammatory pattern in the airways may existwith increased numbers of eosinophils (54). At least 5 studies have found this inmild to moderate COPD exacerbations in airway wall biopsies, and bybroncho-alveolar lavage and sputum induction, although some of these studiesalso included patients with chronic bronchitis without airway obstruction(17;19;27-29).The increase in eosinophils during COPD exacerbations is at leastpartially related to viral infections (55;56). Pathogens are recognised by Toll-likereceptors on epithelial cells, which induce the release of severalpro-inflammatory cytokines (33;57). The eosinophil attracting chemokines“Regulated upon Activation, Normal T-cell Expressed, and Secreted” (RANTES),

26


eotaxin, and interleukin-5 (IL-5) have been reported to be increased duringCOPD exacerbations (29;56;58;59).

Although, as documented above, the eosinophil has been consistently shown tobe associated with COPD exacerbations, many clinicians do not intuitivelyconsider this a relevant cell to target in the treatment of exacerbations. It isinteresting, however, to realise that the eosinophil is the most steroid sensitivecell in the airways and that much of what is achieved with corticosteroids duringexacerbations may be related to effects of steroids on eosinophils (60;61). Instable COPD, it has been shown that higher number of eosinophils correlatewith responsiveness to both oral and inhaled corticosteroids (50;52;62).Additionally, it has been shown recently that both prednisolone and thecombination of inhaled budesonide plus formoterol suppress sputumeosinophilia during COPD exacerbations (11).Whether this steroid-induced decrease in eosinophils has clinical benefit has notdirectly been proven. However, a decrease of soluble interleukin-5 receptoralpha in the resolution phase of a virus-induced exacerbation has been relatedto an increase in forced expiratory flow in 1 second (FEV1), suggesting that sucha relationship may exist (56).

Not only the eosinophils themselves, but also their products such as eosinophilcationic protein are increased in sputum and in serum during COPDexacerbations (29;63). Eosinophil cationic protein among other effects causestissue damage and tissue remodelling in in vitro studies (64). This could explainat least part of the association between exacerbation frequency and excessdecline in lung function (2).

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Table 2: Studies reporting increased airway eosinophils during exacerbations compared to stablephase.

Study Number ofpatients instable phase/exacerbation


Number ofsputumeosinophils instable phase/exacerbation

% sputumeosinophils ofin stablephase/exacerbation

Method

Fujimoto et al(29)

30 / 30 52.9 / 40.6 0.1 / 1.3x106/grama

1.7 / 6.7a Sputuminduction

Bathoorn et al(27)

39 / 39 61 / 51 0.1 / 0.4x106/mLa


Mercer et al(28)

19 / 12 n.r./37.6 0.01 / 0.07x106/grama


Balbi et al(137)

8 / 5 71 / 64 1.9 / 6.7x103/mLa

n.r./n.r. a BAL

Papi et al (17) 15 / 15subgroup viralexacerbations

n.r./n.r. 0.9 / 3.5x106/grama

n.r./n.r. Sputuminduction

_______________________________________________________________________________a: p <0.05n.r.: data not reported

Lymphocytes

Stable diseaseLymphocytes are thought to play an important role in the development andprogression of COPD. Particularly CD8+ lymphocytes have been intensivelyinvestigated. CD8+ cells are increased in the airway submucosa and inperipheral blood in patients with stable COPD, and the number of CD8+ cells ispositively related to the severity of airflow limitation (65-67). Most CD8+ cells aresupposed to represent cytotoxic memory cells, which are produced after a firstairway infection, and facilitate a faster and more effective response of theimmune system when the next infection occurs. Indeed, in vivo studies showthat more effective airway viral clearance is associated with higher CD8+ cellnumbers. CD8+ cell numbers in the airways remain high for several monthsafter viral infections, and stabilize after 6 months (68-70). The higher CD8+numbers in stable COPD patients may be caused by the occurrence of anairway infection in the preceding few months, or alternatively there is acontinuous low grade infection and the numbers of CD8+ cells reflect the needto protect the lung tissue (71-73).CD4+ cells, the helper T cells which produce pro-inflammatory cytokines, havealso been reported to be increased in the peripheral blood of patients withCOPD, particularly the interferon gamma producing cells (74).Several recent studies have also shown that B-cells are increased in bronchiolarand bronchial walls in the stable phase of COPD (75-77). B-cells play a role inthe humeral immune response, producing antibodies to antigens. Thepathological role of the increased B-cells in COPD is still uncertain. It has beenspeculated that viral airway infections may underlie the rise in B-cells (78;79),but an autoimmune response, perhaps in reaction to cigarette smoke

28


components or extracellular matrix products has also been postulated(75;80;81).

ExacerbationsDuring exacerbations lymphocytes in both induced sputum and tissue biopsiesincrease even further (27-29;82). This could partly be explained by the role oflymphocytes in the clearance of viruses. Despite consistent reports of theinvolvement of lymphocytes in COPD exacerbations, little data has beenpublished on the lymphocyte subpopulations which are involved. In a smallstudy, a CD8 type 2 mediated immune reaction occurred during COPDexacerbations (83). To the best of our knowledge, no information on B-cells inCOPD exacerbations has been published. We have no real insight on whetherthe changes in lymphocytes during exacerbations are a normal appropriate, aninsufficient, or even an inappropriate or excessive response. If the response isinsufficient, higher levels of specific lymphocytes may be more protective.Vaccination might be an intervention to increase the levels of specificlymphocytes. To explore whether vaccinations could be an effective interventionto prevent COPD exacerbations, more insight on the role of the lymphocyte andtheir subpopulations is needed. A large observational trial aiming to assess therole of T-cells in COPD exacerbations, which has started recruiting, will hopefullyprovide more information on this topic (ClinicalTrials.gov Identifier:NCT00281229).In summary, airway infections are a common cause of COPD exacerbations,and as lymphocytes are the regulatory cells of immune response to infections,they could very well be key-players in the increased inflammation in both stableCOPD and in the onset of COPD exacerbations, but the exact mechanisms bywhich the influx of cells is generated, their activation state, and their resultingeffects still need to be elucidated.

Table 3: Studies reporting increased airway lymphocytes during exacerbations compared to stablephase.

Study Number ofpatients instablephase/exacerbation


Number ofsputumlymphocytes instable phase/exacerbation

% sputumlymphocytesin stablephase/exacerbation

Method

Fujimoto et al(29)

30 / 30 52.9 / 40.6 0.2 / 0.7x106/grama


Bathoorn et al(27)

39 / 39 61 / 51 0 / 0.1x106/mLa

0.3 / 0.7 a Sputuminduction

Mercer et al(28)

19 / 12 n.r. / 37.6 0.03 / 0.31x106/grama


Papi et al (17) 64 / 64 49.5 / 39.4 0.04 / 0.11x106/gramb

n.r. Sputuminduction

_______________________________________________________________________________a: p <0.05b: p=0.06N.r.: data not reported

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Macrophages

Smokers with stable COPD have increased numbers of macrophages in airwaytissue compared to chronic bronchitic patients without airflow limitation, orhealthy controls (66). The increase in macrophages is induced by smoking,since cessation of smoking in asymptomatic persons results in a decrease insputum macrophages, and current smoking is positively related to macrophagenumbers in airway submucosa and tissue (84;85).During exacerbations of COPD, large observational studies have not shown asignificant increase in sputum or airway tissue macrophages, neither as apercentage of total cells nor as an increase in absolute cell counts(17;29;82;86). One study has even shown a significant decrease in sputummacrophages as percentage (83). However, it is perhaps too early to concludethat macrophages are not involved during exacerbations. Since macrophagesare responsive to cigarette smoke, a reduction in the number of cigarettessmoked during exacerbations could perhaps mask an increase in macrophagescompared to the stable phase of disease. Future studies analysing theinflammatory changes for non-smokers separately could perhaps give a moredefinitive answer whether macrophages are increased during COPDexacerbation.

Air pollution

Epidemiological studies have shown that air pollution, particularly with fineparticulate matters, can cause of COPD exacerbations (15;87). Air pollution isassociated with increased inflammation in the airways of elderly people, as theyhave higher exhaled nitric oxide levels when airway pollution increases (88).There is a lack of data on cellular airway inflammation caused by airwaypollution induced COPD exacerbations, probably since it is difficult to identifythese exacerbations caused by air pollution singularly, and since air pollutionalso interacts with viruses, resulting in a mixed origin of the exacerbation (89).

Oxidative stress

Oxidative stress is an imbalance between the amount of oxidants and thecapacity of antioxidants to scavenge these radicals. This imbalance originatesfrom either an increased load of oxidants (reactive oxygen and nitrogen speciesand free radicals) and/or by a decreased antioxidant capacity. The increase inoxidants may result from release from inflammatory cells, or caused byincreased inhalation of oxidants in cigarette smoke or polluted air (90-92).Oxidative stress induces Nuclear Factor-kappa B, a transcription factor involvedin upregulating genes of many pro-inflammatory cytokines, resulting in increasedinflammation (93). Thus, inflammation induces oxidative stress and oxidativestress in turn causes increased inflammation.Furthermore, the oxidative stress can be caused by a decreased antioxidantcapacity. Theoretically, a decreased antioxidant capacity might result from poornutritional status, however reports on the effect of food intake on the anti-oxidantcapacity is conflicting (94;95). However, an increased oxidant burden may lead

30


to depletion of antioxidants in epithelial lining fluid in exacerbations of COPD(98). Another possibility could be a dysfunction of antioxidant producingenzymes (96;97).During exacerbations, the antioxidant capacity is decreased, both in blood and inthe airway submucosa (98;99). This decrease in antioxidant capacity is probablycaused by an increased requirement for the scavenging of oxidants, sincehydroxygenperoxidase and 8-isoprostane, both products of reactions withoxygen radicals, are increased during COPD exacerbations in exhaled breathcondensate (39;100;101).Thus, oxidative stress is involved in COPD exacerbations, since there is anincreased load of oxidants and the anti-oxidant capacity is decreased. Whetheroxidative stress is a pathogenical cause of exacerbations, or a consequence ofincreased inhalation of oxidants, or infection with an increased inflammation, cannot be deducted.

Anti-inflammatory therapies

Modification of inflammation during COPD exacerbations is not without potentialrisks, since inflammatory cells play a role in defence against pathogens. Ideallyany modification should improve symptoms and inflammatory damage, withoutincreasing the risk of consequent infections. In vivo models of COPDexacerbations would be useful to test new anti-inflammatory drugs.Unfortunately, successful in vivo models for COPD exacerbations have not yetbeen reported as far as we know.

The only proven successful inflammation modifying therapy for COPDexacerbations thus far is treatment with corticosteroids. Treatment of COPDexacerbations with systemic corticosteroids improves lung function andoxygenation, reduces treatment failures, and shortens the length of hospital stay(60;61;102;103). These beneficial effects are evidence based, however themagnitude of effect is modest. Lung function improves in the first 72 hours oftreatment, but the improvement is not significantly different compared to placebotreatment after 2 weeks suggesting a spontaneous recovery in many patients.The reduction in length of hospitalisation with systemic steroids is 1-2 days(60;61;102). However, these gains with steroid treatment come at a price.Treatment with systemic corticosteroids results in adverse systemic effects,such as hyperglycaemia, insomnia, and weight gain (61;102;104). It has beencalculated that one extra adverse effect occurs for every 6 patients treated withsystemic corticosteroids (10).

To avoid systemic adverse effect of oral steroids, treatment with inhaled steroidsat an increased dose compared to the maintenance dose might be an option.Indeed, in one study treatment of COPD exacerbations with inhaled steroidsimproved lung function compared to placebo treatment, and caused lesssystemic effects than systemic steroid treatment (102). Since maximalbronchodilation and steroids are the current standard treatment of COPDexacerbations, combined long-acting bronchodilators and inhaled steroids is alogical next intervention. A single study has investigated treatment of COPDexacerbations with combined budesonide and formoterol, which resulted in a

31


decrease in sputum eosinophils, and an improvement of symptoms and healthstatus (11). A larger study powered to document improvement in airflowlimitation is under way and investigates whether this combined therapy is aseffective as systemic steroids in the treatment of COPD exacerbations(ClinicalTrials.gov Identifier: NCT00259779).

The data on inflammation that observational studies of COPD exacerbationshave provided, can potentially lead to development of novel inflammationmodifying drugs. Some of these drugs have already been tested in the stablephase of COPD, and could be beneficial in the treatment of exacerbations.Modification of inflammation by leukotriene antagonists has been tested in thetreatment of stable COPD (105;106). Both treatment of stable COPD patientswith montelukast, a leukotriene receptor antagonist, and with BAYx1005 aleukotriene synthesis inhibitor resulted in a reduction in neutrophil numbers insputum. Leukotriene B4 has been shown to be involved in the chemoattractionof neutrophils during exacerbations (39). Therefore, treatment to reduceleukotriene levels might also be beneficial in COPD exacerbations.Phosphodiesterase (PDE)-4 inhibition is an anti-inflammatory mechanism thatinhibits the break down of cyclic adenosine monophosphate in inflammatorycells (107). This results in higher intracellular cyclic adenosine monophosphate,which inactivates pro-inflammatory transcription factors by protein kinase A.Trials in patients with stable COPD showed improvements in lung function,quality of life, and exacerbation rates with this treatment (108;109). A reductionof bronchial wall CD8+ cells and macrophages has been demonstrated in stabledisease, but the effect was rather small (110;111). PDE-4 inhibitors have notbeen tested in the treatment of COPD exacerbations.Tumor necrosis factor-a (TNF-a) is a general pro-inflammatory cytokine, and itslevels are increased in sputum at the onset of COPD exacerbations (42).Inhibition of tumor necrosis factor-a could lead to a reduction in inflammationduring COPD exacerbations. Systemic anti-TNF-a administration has beentested in 22 patients with mild-to moderate COPD, yet in a stable phase ofdisease (112). This study showed no positive effects. Perhaps more severeCOPD, or more specifically COPD exacerbations, would be an appropriateindication for anti-TNF-a treatment.

Inhibition of cytokines involved in the recruitment of eosinophils, such as IL-5,RANTES, and eotaxin could also be a strategy to modify inflammation duringCOPD exacerbations. This has not been studied in COPD so far. In vitro studiesand studies in patients with asthma show beneficial effects of such specificanti-eosinophil therapies, and perhaps these therapies should be tested as atreatment of COPD exacerbations (113-121).

Another pathway to reduce inflammation is inhibition of the mitogen activatedprotein (MAP)-kinase pathway. MAP-kinase pathways are involved in the signaltransduction from an external inflammatory stimulus to an inflammatoryresponse, by activating intracellular transcription factors for pro-inflammatorycytokine gene expression (122). In vivo studies investigating MAP-kinaseinhibitors show reducing effects on neutrophil inflammation in alipopolysaccharide inhalation model, and on eosinophilic inflammation in an

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allergy model (123;124). The first generation MAP-kinase inhibitors hadsignificant side effects in humans. A second generation is in development withfewer side effects (125) and might be introduced in the treatment of airwayinflammatory diseases in the near future, including COPD exacerbations.

Macrolides are well known for their anti-microbial activity. Besides theiranti-microbial activity, macrolides have anti-inflammatory effects. They reducepro-inflammatory cytokine production, and neutrophilic and eosinophilicinflammation (126). Part of these anti-inflammatory effects involve theextra-cellularly regulated protein kinase pathway, a MAP-kinase pathway (127).Macrolide treatment in stable COPD patients induced a reduction in neutrophilicinflammation without improvement in health status or exacerbation rate(128;129). We did not find any reports on the effects of COPD exacerbationswith macrolides compared to other antibiotics in relation to theiranti-inflammatory properties.

Inhibition of MAP-kinase can also be established by inhalation of low dosecarbon monoxide (CO) (130). CO inhalation is usually associated with toxiceffects which occur during exposure to high doses or to long-term low levels. Incontrast, exposure to low dose CO can be cytoprotective (131), and both in vitroand in vivo studies have shown its anti-inflammatory effects on the airways(124;132;133). Inhalation of low dose CO in an ovalbumin-induced allergic invivo model attenuates the eosinophilic inflammation by reducing IL-5 levels, andreduces bronchial hyperresponsiveness to methacholine. Therefore, CO hasbeen postulated to have a potential therapeutic role in pulmonary medicine(134). The effects of inhaled CO have been tested in healthy volunteers, whowere infused with lipopolysaccharide, but CO did not influence plasma TNF-a,IL-6, and IL-8 levels, which were increased by lipopolysaccharide (135). Sincethe predominant effect of CO seems to be a reduction of eosinophils, asthmaand COPD exacerbations could be more appropriate indications. The results ofanti-inflammatory effects of inhaled CO on inflammatory airway diseases willappear in the near future (136).

Conclusions

During COPD exacerbations, there is increased airway wall inflammation, withpathophysiological influx of eosinophils, neutrophils, and lymphocytes. There areno reports of increased macrophages during COPD exacerbations. Althoughlinks have been suggested between the increase in eosinophils and lymphocytesand a viral aetiology of the exacerbation, and between the increase inneutrophils and a bacterial aetiology, these increases in both inflammatory celltypes are not limited to the respective aetiologies and the underlyingmechanisms remain elusive. Reports on increases in lymphocytes during COPDexacerbations are consistent, and they might play a key role in the protectionagainst recurrent infections, which evoke an inflammatory response. There islittle data on the subtypes of lymphocytes involved in the onset of COPDexacerbations, which would be essential to dissect the normal from thepathophysiological immune response, which may be increased eitherexcessively or insufficiently. Studies that document the onset of inflammatory

33


changes in COPD exacerbations should prove useful to developinflammation-modifying interventions.

The only successful inflammation-modifying drugs during exacerbations so farare corticosteroids, but their beneficial effects are modest and steroids do haveimportant side effects. New data suggest that the use of inhaled steroids (incombination with long acting bronchodilators) may be an alternative to systemicsteroids in the treatment of exacerbations with less potential for systemic sideeffects. Whether this is as effective as systemic steroids needs to be assessedin future studies.

Several more specific cytokine or pathway inhibiting drugs are in developmentfor stable COPD. A further step might be to test these drugs also in COPDexacerbations. However, it is also possible that new drugs that specifically targetinflammatory changes pertinent to COPD exacerbations can be developed. Inconclusion, further research is required to fully understand the inflammatorymechanisms in the onset and development of COPD exacerbations. This mightmake inflammatory pathway-specific intervention possible, resulting in a moreeffective treatment of COPD exacerbations with fewer side effects.

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(2) Donaldson GC, Seemungal TA, Bhowmik A, Wedzicha JA. Relationship betweenexacerbation frequency and lung function decline in chronic obstructive pulmonarydisease. Thorax 2002; 57(10):847-852.

(3) Rutten-van Molken MP, Postma MJ, Joore MA, Van Genugten ML, Leidl R, Jager JC.Current and future medical costs of asthma and chronic obstructive pulmonary disease inThe Netherlands. Respir Med 1999; 93(11):779-787.

(4) McGuire A, Irwin DE, Fenn P, Gray A, Anderson P, Lovering A et al. The excess cost ofacute exacerbations of chronic bronchitis in patients aged 45 and older in England andWales. Value Health 2001; 4(5):370-375.



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Chapter 3

Safety of sputum induction during exacerbations ofchronic obstructive pulmonary disease

Erik Bathoorn MD1, Jeroen Liesker MD1, Dirkje Postma MD PhD1,Gerard Koëter MD PhD1, Antoon J.M. van Oosterhout PhD2, HuibA. M. Kerstjens MD PhD1

1 Groningen Research Institute for Asthma and COPD (GRIAC),Department of Pulmonology, and 2 Laboratory of Allergology andPulmonary Diseases, University Medical Center Groningen,University of Groningen, the Netherlands.

Published: Chest 2007; 131:432-438

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Abstract

Sputum induction is considered to be a safe tool to assess airway inflammationin patients with stable chronic obstructive pulmonary disease (COPD), but little isknown about its safety during exacerbations. We therefore assessed the safetyof sputum induction during COPD exacerbations.Sputum induction data of 44 COPD patients was assessed both in their stablephase and during exacerbation. Median forced expiratory flow in one second(FEV1) during stable phase and exacerbation were 61 (49-74) % and 51 (45-60)% predicted, respectively.The median (interquartile range) decrease in FEV1 with sputum induction duringan exacerbation was 0.27 L (0.17-0.40) versus 0.28 L (0.22 - 0.44) during thestable phase (p=0.03). The patients sustained the associated dyspnea well; noother adverse events occurred. All FEV1 values returned to within 90% of theirinitial value within 30 minutes. A larger decrease in FEV1 due to sputuminduction during an exacerbation was associated with the following parametersin the stable phase of disease: lower total sputum cell count (r=-0.37, p=0.01),higher percentage of eosinophils (r=0.33, p=0.04), and a larger decrease inFEV1 after sputum induction (r=0.39, p=0.03). In a multivariate analysis, the onlyindependent association was with the larger decrease in FEV1 in the stablephase.We conclude that sputum induction can be safely carried out in patients withmild to moderate COPD who experience an exacerbation, and this occurs withno greater risk than in stable COPD.

This study has been registered at http://www.clinicaltrials.gov, ID:NCT00239278.

List of abbreviations:COPD= chronic obstructive pulmonary diseaseFEV1= forced expiratory flow in one secondkg= kilogramL=literm= meterµg= microgrammin= minutepred= predictedsec= secondSI= sputum inductionVC= slow inspiratory vital capacity

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Safety of sputum induction during exacerbations of chronic obstructive pulmonary disease

Introduction

Sputum cell differential counts reflect the level of inflammation in the airways inpatients with chronic obstructive pulmonary disease (COPD) 1-3 and aretherefore increasingly assessed in both research and clinical settings. Since notall patients produce sputum spontaneously, the coughing up of sputum isroutinely facilitated by inhalation of a saline solution; hypertonic saline is themost frequently used. For methodological reasons, the induced method isapplied both in patients with and without spontaneous sputum production 4.Inhalation of both isotonic and certainly of hypertonic saline causes abronchoconstrictive response in many patients with COPD. So far, only a fewstudies have evaluated the safety of sputum induction in these patients, and allassessed safety during a stable phase of the disease. Average decreases inforced expiratory volume in one second (FEV1) with sputum induction rangedfrom 0.12 to 0.36 liter in different studies carried out in stable COPD patients 5-9.Since the decrease in FEV1 is generally transient and more severe adverseeffects do not occur, it has been put forward that sputum induction is safe evenin patients with moderate to severe COPD, but should be monitored carefullysince sometimes severe bronchoconstrictive reactions can occur 6;9. TheEuropean Respiratory Society Task Force regarding the safety of sputuminduction concluded in 2002 that "sputum induction has been used safely insubjects with severe COPD, but there have been no systematic studiesaddressing safety issues in this patient category" 2.To the best of our knowledge, no studies have reported data about the feasibilityof sputum induction during exacerbations of COPD. We wished to carry outsputum inductions during an exacerbation. However, we anticipated that thismight induce very low FEV1 values, given the already reduced FEV1 duringexacerbations. Therefore we applied a modified protocol used in severe asthmaexacerbations by Pizzichini and co-workers, to assess safety of sputuminduction during an exacerbation 10. We evaluated whether it is safe to performsputum induction during an exacerbation of COPD. We also investigatedwhether it is possible to predict the decrease in FEV1 due to inhalation ofnebulised saline during exacerbations from the patient characteristics, fromdecreases in FEV1 by the induction procedure during stable phase of thedisease, or from inflammatory cells in induced sputum during stable phase ofCOPD.

Methods

PatientsData were obtained from patients participating in an ongoing clinical trial duringthe period before randomisation. The inclusion criteria were a diagnosis ofCOPD, age above 40 years, a postbronchodilator FEV1 below 85% predicted(pred.) but above 0.7 liters (L), and an FEV1/slow inspiratory vital capacity (VC)

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below predicted normal (<88% pred. in men and <89% pred. in women) afterbronchodilation. Patients were not allowed to use oral corticosteroids, longacting anticholinergics, beta-blockers, or oxygen therapy, and to have a historyof asthma or significant other diseases that could influence the results of thestudy. The medical ethics committee approved the study. A written informedconsent was obtained from all patients prior to the study.

Study designAt the inclusion visit, inhaled corticosteroids, when used, were discontinued,whereafter the subjects had to be stable for 2 months. At the second visit, 2months later, spirometry was performed followed by a sputum induction. Theresults of these latter measurements are used as baseline, stable phase values.From the second visit, all long-acting beta2-sympaticomimetics were withdrawn.After this, patients were asked to contact the research doctor at any time of theday or night to report any deterioration in symptoms for which they wouldnormally contact either their primary care physician or their pulmonologist. Anexacerbation was defined according to Davies 11: a history of increasedbreathlessness and at least two of the following symptoms for =24 hours:increased cough frequency or severity, increased sputum volume or purulence,and increased wheeze. During exacerbation, the postbronchodilator FEV1 had tobe < 70% of predicted. Patients were not accepted for sputum inductiontreatment if the FEV1 was < 0.8 liter and the arterial oxygen pressure was below8.0 kPa.

MeasurementsIn a stable phase of COPD and during an exacerbation, sputum induction, andlung function measurements were performed. FEV1 and VC were measuredaccording to the guidelines of the European Respiratory Society 12.In view of our concern of performing sputum induction safely in patients with alow FEV1 we used 2 methods using different tonicity of saline dependent on thedegree of bronchoconstriction. We adapted a protocol by Pizzichini andco-workers in asthma which starts with isotonic saline in shorter exposition timesand gradually increases tonicity and exposition in subjects with a lower FEV1leading to closer monitoring of the decrease in FEV1 (figure 1). FEV1 wasmeasured 20 minutes after inhalation of 400µg salbutamol. 1) If the FEV1 was above 1.5 L, sputum induction was performed using 4.5%hypertonic saline for 3 times 5 minutes (regular protocol). 2) If the FEV1 was below 1.5 L, sputum induction was started using 0.9%saline and the tonicity of the saline nebulisation was gradually increased asdepicted in figure 1 (cautious protocol).As much as sustainable, patients were encouraged to accomplish all the stepsof the entire procedure, also if sufficient sputum has already been produced.The subjects inhaled the saline from an ultrasonic nebuliser (DeVillbiss Neb2000, Somerset, Pennsylvania, United States of America) with an output of 1.5mL/minute.

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Whole sputum samples were processed within 120 minutes as describedpreviously 1. Cytospins were prepared and cell-free supernatant was collectedand stored in aliquots at -80 C pending analyses of soluble mediators.Differential cell counts were counted on May Grünwald Giemsa stainedcytospins in a blinded fashion 13. Cell counts were expressed as percentage ofnon-squamous cells. A sputum sample was considered inadequate when thepercentage of squamous cells was >80%.

Figure 1: Sputum induction protocol . * After each step FEV1 is measured. If the decrease in FEV1is more than 20%, the sputum induction is completely stopped. If the decrease in FEV1 is 10-20%of postsalbutamol FEV1, patients receive 200 µg salbutamol. Ten minutes after inhalation ofsalbutamol, the FEV1 is measured again. If the decrease in FEV1 is still more than 10%, thesputum induction is stopped. After each step the patients are asked to cough up sputum. As muchas sustainable, patients are encouraged to accomplish all the steps of the entire procedure, also ifsufficient sputum has already been produced. FEV1: Forced expiratory volume in one second; L= liters;µg= micrograms; sec= second; min=minute

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Statistical methodsData in tables are presented as medians (interquartile range). Changes in FEV1during sputum induction are expressed as decreases in liters (larger numberssignifying larger decreases). Differences in decreases in FEV1 during sputuminduction between the exacerbation phase and the stable phase were analyzedby paired sample T-test. Baseline data, such as age, spirometric indices, andparameters from the baseline (stable phase) sputum induction were analyzed fortheir correlations with the decrease in FEV1 by the sputum induction during anexacerbation. Continuous variables were correlated with the maximal decreasein FEV1 during sputum induction by Pearson’s correlation test, after testing fornormal distribution. Parameters which were not normally distributed were logtransformed. Parameters which were not normally distributed after logtransformation were correlated using Spearman’s correlation test. Categoricalvariables were analyzed using independent samples T-tests for the differencesin maximal decrease in FEV1. The parameters showing a significant correlation,and the protocol used were entered in a multiple linear regression model.P-values <0.05 were considered significant. All data were analyzed with theSPSS statistical package for Windows, version 10 (SPSS inc, Chicago, Illinois).

Results

Subject characteristicsOne-hundred-and-fourteen patients with COPD were recruited. Forty-fivesubjects experienced an exacerbation during the study. One patient's FEV1data during sputum induction has not been recorded. The data of the remaining44 patients were used in the analyses (table 1). In stable phase, 91% of thesputum samples had an assessable cytospin, and during an exacerbation 93%.

Decrease in FEV1 during sputum induction and other adverse eventsThe changes in FEV1 during sputum induction in the stable and exacerbationphase are presented in table 2 and figure 2. A decrease in FEV1 of 10-20%during sputum induction occurred in 41% of patients during an exacerbation,39% had a decrease in FEV1 of >20% compared with the initial values. Thedecrease in FEV1 during sputum induction did not differ significantly between theregular and the cautious protocol, i.e. when the FEV1 was above or below 1.5 L.There was a slightly but significantly smaller decrease in FEV1 with sputuminduction during an exacerbation (median decrease 0.27 L versus stable phase0.28 L; figure 2). The difference between the stable and exacerbation phase inthe induced decrease in FEV1 was not different between patients whounderwent the cautious and the regular protocol (see figure 1). The lowest FEV1reached during sputum induction was not significantly different in patients duringthe stable phase measurement compared to during an exacerbation (table 2).Figure 3 shows the cumulative % of patients induced by the cautious protocolwho fulfilled each step of the protocol. The patients had the same number of

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protocol steps during exacerbations as during stable phase. Although anoccasional large fall did occur (one patient had a maximal decrease of 700 mL),all patients sustained the procedure and the associated increase in dyspneavery well. All FEV1 values returned to within 90% of the initial (post-salbutamol)value within 30 minutes. No further measures were needed. No other adverseevents occurred during sputum inductions.

Table 1. Patient characteristics_______________________________________________________________________________

n=44Sex (male/female) 36/8Age (years)* 64 (58-71)Smoking status, current/ex 21/23Packyears* 38 (26-49)Body mass index (kg/m2)* 25 (24-28)FEV1, (% pred)* inclusion 61 (49-74)FEV1, (L)* inclusion 1.84 (1.42-2.25)FEV1, (% pred)* exacerbation 51 (45-60)FEV1, (L)* exacerbation 1.58 (1.23-1.94)FEV1/VC% pred.* inclusion 45 (38-54)FEV1/VC% pred.* exacerbation 37 (32-46)Reversibility (% pred)* 9 (5-11)Sputum total cells x106/mL in stable phase 8.2 (2.2-18.7)

Sputum neutrophils % in stable phase 72 (65-80)Sputum eosinophils % in stable phase 2.7 (0.8-5.8)Sputum lymphocytes % in stable phase 0.3 (0.2-1.3)Sputum macrophages % in stable phase 21 (14-25)

_______________________________________________________________________________

*median (interquartile range)FEV1= Forced expiratory volume in one secondVC= slow inspiratory vital capacityPred=predictedkg= kilogramm= meterL= liter

.

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Figure 2: Differences in decrease in FEV1 due to sputum induction in stable phase and inexacerbation of COPD. In the total group, the decrease in FEV1 by sputum inductions was minutelylower during exacerbation compared to the decrease in their stable phase (mean decreases 0.27 Lversus 0.28 L respectively; p=0.03). FEV1= Forced expiratory volume in one second; L= Liters

Variables associated with the decrease in FEV1 by sputum induction during anexacerbation

Correlation coefficients and p-values of the association between the decrease inFEV1 at the exacerbation visit with the predefined parameters in the stablephase of disease are presented in table 3. A larger decrease in FEV1 during anexacerbation correlated significantly with a larger decrease in FEV1 duringsputum induction at stable phase, a lower total cell count, and a highereosinophil% in induced sputum at stable phase. There were no significantdifferences in the decrease of FEV1 according to divisions by smoking status,sex, and protocol used for sputum induction. The variables age, sputuminduction protocol, decrease in FEV1 due to sputum induction in stable phase,total cell count, and eosinophil% in induced sputum in the stable phase of COPDwere entered in a multiple regression model. The severity of the decrease inFEV1 during sputum induction in the stable phase was the only independentpredictor of a larger decrease in FEV1 during sputum induction the exacerbation.Figure 4 shows the correlation between the decrease in FEV1 by sputuminduction during the stable phase and during exacerbation (r=0.44, p=0.03)

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Table 2. Decrease in FEV1 during sputum induction. Data were analysed with paired T-test fordifference between decrease in FEV1 by sputum induction in a stable phase of disease and duringexacerbation.

Stable ExacerbationInduction protocol † Total Cautious Regular Total Cautious RegularNumber of patients 44 16 28 44 19 25Decrease in FEV1with sputum induction (L)*

0.28(0.22-0.44)

0.26(0.20-0.28)

0.36(0.24-0.50)

0.27 ‡(0.17-0.40)

0.23(0.20-0.30)

0.31(0.10-0.45)

Decrease in FEV1with sputum induction (% ofpost-salbutamolFEV1) *

20(15-25)

21(19-23)

20(13-28)

19(13-25)

20(18-25)

17(6-24)

number with decreasein FEV1 10-20%

13 5 8 18 9 9

number with decreasein FEV1 >20%

25 11 14 17 9 8

Lowest FEV1 reached(L)*

1.2(1.0-1.6)

1.0(0.8-1.0)

1.5(1.2-1.8)

1.2(0.9-1.7)

0.9(0.8-1.1)

1.6(1.3-1.9)

_______________________________________________________________________________* median (interquartile range) ‡: p<0.05† Cautious protocol in patients with post-bronchodilatorFEV1 below 1.5 L; regular protocol in patients with FEV1 above 1.5 L. FEV1= Forced expiratoryvolume in one second L= liter

Table 3: Correlations with decrease in FEV1 by sputum induction during exacerbation._______________________________________________________________________________

Pearson’s r p-valueAge (years) -0.30 0.05Packyears -0.05 0.77Body mass index (kg/m2) -0.24 0.11FEV1, (% pred) exacerbation 0.21 0.17FEV1/VC exacerbation 0.21 0.18Bronchodilator response (% pred) -0.08 0.62Decrease in FEV1 by sputum induction instable phase

0.44 0.03

Sputum total cell count stable -0.37 0.01Sputum neutrophil % stable -0.11 0.51Sputum macrophage % stable -0.13 0.94Sputum eosinophil % stable 0.33 0.04Sputum weight stable -0.21 0.18

_______________________________________________________________________________

FEV1= Forced expiratory volume in one secondVC= slow inspiratory vital capacitypred=predictedkg= kilogramsm= meters

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Figure 3: The cumulative % of patients who were induced by the cautious protocol in both stablephase and at exacerbation (n=14), fulfilling each step of the cautious protocol, starting with 3 stepsof isotonic saline (see figure 1.). The geometric mean number of steps fulfilled were 5.5 in stablephase of disease and 5.6 during exacerbation (p=0.97).

Figure 4: Relation between decrease in FEV1 during sputum induction in stable phase andexacerbation (r=0.44, p=0.03), marked by the protocol used during sputum induction atexacerbation (see figure1 for the sputum induction protocol). There was no significant difference inthe decreases in FEV1 during sputum inductions between the 2 protocols used. FEV1= Forcedexpiratory volume in one second; SI= Sputum induction; L= Liters

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Discussion

Our study shows that sputum inductions can be performed safely duringexacerbations of COPD. Considerable decreases in FEV1 can occur, but theyare sustained well. The decreases in FEV1 by sputum induction during anexacerbation are of similar absolute magnitude or even smaller than in thestable phase. Monovariate analysis showed that a larger decrease in FEV1 dueto sputum inductions during exacerbations can be predicted by a largerdecrease in FEV1, a lower sputum total cell count, and a higher eosinopihil% inthe induced sputum differential count during the stable phase of COPD. Thesole independent predictor of the fall in FEV1 during sputum induction in COPDpatients with an exacerbation was the decrease in FEV1 during sputuminduction at stable phase.

Sputum induction has been shown previously to be a safe procedure duringstable phase asthma 14, during exacerbations of asthma 15, and in patients withstable COPD, even in more severe disease 16. We now show additionally thatsputum can be induced safely during exacerbations of patients with mild tomoderate COPD (i.e., those exacerbations that would have otherwise beentreated at home with a course of prednisolone with or without antibiotics).

A few studies already used sputum induction during COPD exacerbations 17-19,relying on experiences with sputum induction in patients with stable COPD 16.Noteworthy, all studies thus far performing sputum induction in COPDexacerbations used different protocols. The studies started either with isotonic orhypertonic saline varying from 3%-4.5% saline, finished the whole inductionprotocol or stopped after production of 2 mL sputum, or used spontaneoussputum if serious consequences from the sputum induction were expected.Unfortunately none of these studies reported evaluation of the safety of sputuminductions (by reporting decreases in FEV1 or otherwise), which would havebeen very useful for the development of a universal protocol for sputuminductions during COPD exacerbations.

In view of our concern of performing sputum induction safely in patients with alow FEV1 and even more so when patients experienced an exacerbation, weadapted a protocol by Pizzichini and coworkers 10. The protocol starts withisotonic saline in shorter exposition times and gradually increases tonicity andexposition in subjects with a lower FEV1. With this protocol, the falls in FEV1were considerable in some of our individuals (up to 700mL), but all patientsrecovered quickly with salbutamol and time. No clinically relevant adverseeffects were encountered. Since we did not compare both protocols in the samepatients with a lower FEV1, it is conceivable that no major problems would havebeen encountered also with the regular protocol that we now used only inpatients with an FEV1>1.5. However, because of safety considerations, we didnot dare do the comparison but adhered to this protocol that is cautious indesign.

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There are some potential drawbacks to our cautious protocol. First of all, it takesmuch longer to perform the induction, both for the patient and the technician.The average time with the regular protocol in patients with an FEV1 above 1.5 Lis about 35 minutes. This may increase to 75 minutes with the cautious protocolin patients with an FEV1 below 1.5 L. Secondly, if patients have an FEV1 around1.5 L, they could, with repeated sputum inductions in a study and strictadherence tot the cut-off values, at one day be subjected to the regular protocoland at another day to the cautious protocol. This would imply that the durationand saline concentrations used during an exacerbation are not the same asduring the stable phase of disease, which conceivably could affect the results ofthe sputum induction. Taube et al compared sputum induction with 0.9% or 3%saline in patients with COPD and found no significant differences in total anddifferential cell counts in the induced sputum 9. Holz et al demonstrated inasthmatic and healthy subjects that the sputum neutrophil% decreased andmacrophage% increased in the samples of three sequential periods of 10minutes sputum induction. They advocated a protocol with a standardizedduration of the procedure 20 which is why we fulfilled all steps of the protocol asmuch as possible instead of stopping when sufficient sputum has beencollected. Belda et al investigated the effect of nebulisation output and durationon the cell counts and fluid-phase measures in asthmatic patients. Theyconcluded that the samples of the longer duration of sputum induction werelower in sputum weight, neutrophil and eosinophil%, eosinophil cationic proteinand interleukin-8 levels, and higher in sputum macrophage counts 21;22. It is forsafety reasons that we have used the cautious protocol in patients with a lowFEV1. In patients who have an FEV1 just above 1.5 liter in the stable phase andwho are therefore at risk to obtain an FEV1 below 1.5 liter during anexacerbation, it is probably better to start with the cautious protocol from thebeginning of the study onwards, when measurements are planned to beperformed per protocol in an exacerbation phase as well.

In the present study, a significant correlation was found between the decrease inFEV1 during sputum induction during an exacerbation and a larger decrease inFEV1 during induction at stable phase. Large bronchoconstrictive reactions tonebulised saline seem to occur irrespective of the phase of disease (stable orexacerbation). Several previous studies investigated predictors of abronchoconstrictive reaction to nebulised saline in subjects with COPD. Asmaller reversibility to beta2 agonists was found to be associated with a largerfall in FEV1 8. Furthermore a larger decrease in FEV1 during sputum inductioncorrelated with a higher concentration of saline used, higher decrease in peakflow with sputum induction, and higher histamine levels in sputum 5;6;9. We didnot find a correlation with bronchodilator response, or concentration of saline,possibly due to the fact that we used two different protocols for sputuminduction. Our study was not designed to compare decreases of FEV1 inducedby different concentrations of saline, so we might have missed this association.We performed an additional analysis to study if the patients with an eosinophilicexacerbation have a larger decrease in FEV1 due to inhaled saline. In 30% of

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the patients, the sputum eosinophil% was above 3.5% at exacerbation. Therewas no significant difference in the decrease in FEV1 due to inhaled salinebetween the patients with higher and lower eosinophil% at exacerbation.However, we can not rule out the role of eosinophils in the bronchoconstrictivereaction to saline, since the group with higher eosinophil% might be the oneswith a larger initial response to the pretest salbutamol 23.

Our data shows that sputum induction can be performed safely duringexacerbations of COPD. With a cautious protocol that consumes some moretime, the decreases in FEV1 during sputum induction are not larger than duringa stable phase of COPD and pose no major clinical adverse effects. Since alarger decrease in FEV1 during sputum induction in COPD patients experiencingan exacerbation is associated with a higher decrease in FEV1 during sputuminduction at stable phase of disease, it is probably safer to start with a lowconcentration of saline sputum induction in patients now experiencing anexacerbation and who had a large decrease in FEV1 during sputum induction atstable phase. We conclude that sputum can be safely induced in patients withmild to moderate COPD who experience an exacerbation, without a greater riskthan in stable COPD.

Acknowledgements

The authors thank Ibolya Sloots, Dorothea de Reus, and Brigitte Dijkhuizen, forall sputum measurements, the lung function department for the many lungfunction measurements, and prof. F. E. Hargreave for his help with thedevelopment of the sputum induction method.

References

(1) Rutgers SR, Timens W, Kaufmann HF et al. Comparison of induced sputum withbronchial wash, bronchoalveolar lavage and bronchial biopsies in COPD. Eur Respir J2000; 15(1):109-115.

(2) Pizzichini E, Pizzichini MM, Leigh R et al. Safety of sputum induction. Eur Respir J Suppl2002; 37:9s-18s.

(3) Jayaram L, Parameswaran K, Sears MR et al. Induced sputum cell counts: theirusefulness in clinical practice. Eur Respir J 2000; 16(1):150-158.

(4) Bhowmik A, Seemungal TA, Sapsford RJ et al. Comparison of spontaneous and inducedsputum for investigation of airway inflammation in chronic obstructive pulmonary disease.Thorax 1998; 53(11):953-956.

(5) Vlachos-Mayer H, Leigh R, Sharon RF et al. Success and safety of sputum induction inthe clinical setting. Eur Respir J 2000; 16(5):997-1000.

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(6) Sutherland ER, Pak J, Langmack EL et al. Safety of sputum induction inmoderate-to-severe chronic obstructive pulmonary disease. Respir Med 2002;96(7):482-486.

(7) Brightling CE, Monterio W, Green RH et al. Induced sputum and other outcomemeasures in chronic obstructive pulmonary disease: safety and repeatability. Respir Med2001; 95(12):999-1002.

(8) Rytila PH, Lindqvist AE, Laitinen LA. Safety of sputum induction in chronic obstructivepulmonary disease. Eur Respir J 2000; 15(6):1116-1119.

(9) Taube C, Holz O, Mucke M et al. Airway response to inhaled hypertonic saline in patientswith moderate to severe chronic obstructive pulmonary disease. Am J Respir Crit CareMed 2001; 164(10 Pt 1):1810-1815.

(10) Pizzichini MM, Pizzichini E, Clelland L et al. Sputum in severe exacerbations of asthma:kinetics of inflammatory indices after prednisone treatment. Am J Respir Crit Care Med1997; 155(5):1501-1508.


(12) Quanjer PH, Tammeling GJ, Cotes JE et al. Lung volumes and forced ventilatory flows.Report Working Party Standardization of Lung Function Tests, European Community forSteel and Coal. Official Statement of the European Respiratory Society. Eur Respir JSuppl 1993; 16:5-40.

(13) Meijer RJ, Kerstjens HA, Arends LR et al. Effects of inhaled fluticasone and oralprednisolone on clinical and inflammatory parameters in patients with asthma. Thorax1999; 54(10):894-899.

(14) Fahy JV, Boushey HA, Lazarus SC et al. Safety and reproducibility of sputum induction inasthmatic subjects in a multicenter study. Am J Respir Crit Care Med 2001;163(6):1470-1475.

(15) Twaddell SH, Gibson PG, Carty K et al. Assessment of airway inflammation in childrenwith acute asthma using induced sputum. Eur Respir J 1996; 9(10):2104-2108.

(16) Bhowmik A, Seemungal TA, Sapsford RJ et al. Comparison of spontaneous and inducedsputum for investigation of airway inflammation in chronic obstructive pulmonary disease.Thorax 1998; 53(11):953-956.

(17) Aaron SD, Angel JB, Lunau M et al. Granulocyte inflammatory markers and airwayinfection during acute exacerbation of chronic obstructive pulmonary disease. Am JRespir Crit Care Med 2001; 163(2):349-355.

(18) Bhowmik A, Seemungal TA, Sapsford RJ et al. Relation of sputum inflammatory markersto symptoms and lung function changes in COPD exacerbations. Thorax 2000;55(2):114-120.

(19) Fujimoto K, Yasuo M, Urushibata K et al. Airway inflammation during stable and acutelyexacerbated chronic obstructive pulmonary disease. Eur Respir J 2005; 25(4):640-646.

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(20) Holz O, Jorres RA, Koschyk S et al. Changes in sputum composition during sputuminduction in healthy and asthmatic subjects. Clin Exp Allergy 1998; 28(3):284-292.

(21) Belda J, Hussack P, Dolovich M et al. Sputum induction: effect of nebulizer output andinhalation time on cell counts and fluid-phase measures. Clin Exp Allergy 2001;31(11):1740-1744.

(22) Kelly MG, Brown V, Martin SL et al. Comparison of sputum induction using high-outputand low-output ultrasonic nebulizers in normal subjects and patients with COPD. Chest2002; 122(3):955-959.

(23) Papi A, Romagnoli M, Baraldo S et al. Partial reversibility of airflow limitation andincreased exhaled NO and sputum eosinophilia in chronic obstructive pulmonary disease.Am J Respir Crit Care Med 2000; 162(5):1773-1777.

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Chapter 4

Inhaled steroid cessation in stable COPD increasessputum eosinophils and deteriorates lung function

Jeroen J.W. Liesker1, Erik Bathoorn1, Huib A.M. Kerstjens1,Antoon J.M. van Oosterhout2, Gerard H. Koëter1, Dirkje S.Postma11Department of Pulmonology and Tuberculosis, University MedicalCenter Groningen, University of Groningen, Groningen, TheNetherlands. 2Laboratory of Allergology & Pulmonary Diseases,University Medical Center Groningen, University of Groningen,Groningen, The Netherlands

Submitted

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Abstract

Rationale: According to current guidelines, patients with mild COPD should notuse inhaled corticosteroids. However, many patients already using inhaledcorticosteroids can not stop them without a flare up of disease. The reasonsbeyond this are unclear.Objective: To investigate the effects of two-month inhaled corticosteroidwithdrawal on airway inflammation, lung function and health status in COPDpatients with stable disease.Methods and Subjects: Sputum, lung function, and health status wereassessed before and two months after inhaled corticosteroid withdrawal in 56COPD patients (mean age 63 years, mean FEV1 63%predicted).Results: Median percentage sputum eosinophils increased from 1.5% to 2.3%(p=0.003) after two-month inhaled corticosteroid withdrawal. Parameters ofairway obstruction and hyperinflation deteriorated during the same period, e.g.median FEV1 from 64 to 56 %predicted, p<0.0001, median intra-thoracal gasvolume expressed as percentage of total lung capacity from 108 to 109%predicted, p=0.042. Furthermore, mRNA levels of RANTES (regulated onactivation, normal T-cell expressed and secreted), Interleukin-13, Tumor growthfactor-ß, and Tumor necrosis factor-a increased significantly. The change insputum eosinophils correlated significantly with the change in airway obstructionexpressed as FEV1 and FEV1/inspiratory vital capacity (rho = - 0.31, p=0.020and rho= - 0.47, p<0.0001 respectively).Conclusions: Even when patients remain clinically stable, two-month inhaledcorticosteroid withdrawal leads to increased sputum inflammation, andparticularly eosinophils. This increase is associated with a deterioration of airwayobstruction and hyperinflation.

Introduction

Maintenance therapy with inhaled corticosteroids (ICS) in patients with COPDhas been extensively debated in the past decennia and it is still controversial 1-3.On the one hand, it is now well established that ICS do not slow down lungfunction decline in patients with COPD 2. On the other hand, ICS reduce theexacerbation rate in patients with COPD, certainly in GOLD (the Global Initiativefor chronic Obstructive Lung Disease) stage III/IV 3. In clinical practice, manypatients with stage I/II COPD also use ICS 4. Some patients have clear benefitsof ICS treatment suggesting that they should receive ICS. However, patientswho do not benefit should stop ICS according to the GOLD guidelines. Thisdiscontinuation should be monitored carefully since a randomized controlled trialdemonstrated that patients who already use ICS can not sustain stopping them,as exemplified by a rapid onset and higher recurrence rate of exacerbations anda decline in quality of life after discontinuation 5. Wouters et al furthermoreshowed a decrease in lung function and quality of life with an increase inexacerbation frequency after 1 year discontinuation 6. This deterioration isprobably not similar in every patient. Therefore, it would be of value to know

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Inhaled steroid cessation in COPD increases sputum eosinophils and deteriorates lung function

which patients with long-term ICS use can stop ICS and which should continuethis maintenance treatment.

The beneficial effects of ICS in COPD are thought to result from theiranti-inflammatory profile, which has been supported by recent studies 7;8.Therefore, it is tempting to speculate that clinical deterioration after ICSwithdrawal is based on an increase in airway inflammation. The abovementioned discontinuation studies 5;6 did not investigate whether worsening ofthe disease after withdrawal of ICS was accompanied by an increase in airwayinflammation.

The current study addresses the question which is the underlying mechanism ofclinical deterioration after inhaled corticosteroid withdrawal in patients with mildto moderately severe COPD. We hypothesised that discontinuation of ICS inpatients with stable COPD increases airway inflammation, even when it does notlead to a full blown exacerbation. Therefore, we analysed the effects of inhaledcorticosteroid withdrawal on airway inflammation, lung function, and healthstatus by comparing two stable phases, i.e. while still on ICS therapy and twomonths after ICS withdrawal.

Methods

SubjectsCOPD patients were included if on ICS maintenance therapy, age >40 years,post-bronchodilator forced expiratory volume in the first second (FEV1)<85%predicted (pred) but >0.7 liters, and post-bronchodilator FEV1/inspiratoryvital capacity (IVC) <predicted (<88%pred in men and <89%pred in women).Patients with an asthma history long-term oxygen therapy, or significant otherdisease possibly influencing the results of the study were excluded. Oralcorticosteroids were not allowed from 4 weeks before the inclusion visit. Allpatients signed written informed consent and the local medical ethics committeeapproved the study.

Study designHere, we present the analyses of the first two visits of a longer study. Patientdata were collected before and after two-month ICS withdrawal. In case of anexacerbation, visit two was postponed for at least two months after treatment ofthe exacerbation (n=4). The randomized controlled trial from visit 3 onwards willbe reported separately 9.At both visits, history taking and physical examination were performed.Afterwards lung function was performed followed by sputum induction, healthstatus questionnaires, blood, and urine sampling.

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Measurements

Lung function and health statusAt both visits, spirometry and body plethysmography (JaegerPneumotachograph /Masterscreen body, VIASYS Healthcare Inc,Conshohocken, USA) after inhalation of 400 µg salbutamol were performedaccording to ERS standards 10. At the end of both visits, the Chronic RespiratoryQuestionnaire (CRQ) 11 and the Clinical COPD Questionnaire (CCQ) 12 wererecorded.

Sputum induction and processingSputum was induced with modifications according to Hargreave when FEV1 wasbelow 1.5 liters 13. The whole sputum sample was processed (see online datasupplement). Soluble mediators were measured in sputum supernatant byELISA: leukotriene-B4 (LTB4), eosinophilic cationic protein (ECP), andmyeloperoxidase (homemade) (see online data supplement).

Sputum cytokine mRNA expressionMessenger ribonucleic acid (mRNA) was harvested and isolated from 1 x 106

non-squamous sputum cells (see online data supplement). Expression ofcytokine mRNA for haemoxygenase -1 (HO-1), tumor necrosis factor alpha(TNF-a), regulated on activation, normal T-cell expressed and secreted(RANTES) , interleukin 5 (IL-5), IL-10, IL-12, IL-13, transforming growth factor-(TGF- ), interferon- (IFN- ), and 2-microglobulin were analysed byquantitative real-time PCR (see online data supplement). Cytokine geneexpression was normalised to 2-microglobulin expression. The mRNAquantification is expressed in threshold cycle (Ct) values, which is the number ofamplification cycles to reach a detectable mRNA amount (i.e. lower Ct-valuescorrespond with higher mRNA expression). These values were available for 36subjects, because it was added to the protocol later.

Blood and urine analysesMethods for blood differential cell counts, C-reactive protein (CRP), solubleintercellular adhesion molecule (sICAM), and urine desmosine are presented inthe online data supplement.

Statistics

Baseline characteristics are presented as means (+/- standard deviations).Differences over 2 months of corticosteroid withdrawal are presented asmedians (interquartile range), and analysed by Mann-Whitney-U tests. Sinceparticularly sputum eosinophil percentage and lung function deteriorated overtwo months, correlates of these changes with other parameters of inflammation,lung function, and health status were analysed by Spearman correlation-tests. Pvalues =0.05 were considered significant.

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Results

Baseline characteristics

Seventy-five subjects were screened, 56 were included in this analysis. Theirbaseline characteristics are presented in table 1. Reasons for exclusion werepersistent instability after ICS discontinuation (n=9), withdrawal (n=1), or inabilityto produce an adequate sputum sample at visit 1 (n=7) or visit 2 (n=2). Themean (sd) daily dose of inhaled corticosteroids at visit 1 was 817 (328) µg.

Table 1: Baseline characteristics*_______________________________________________________________________________

n=56Gender (m/f) 44/12Actual smoking (yes/no) 25/31

mean s.d.Age (yrs) 63.3 8.1Packyears 44.3 24.0BMI (kg/m2) 26.4 4.0Inhaled corticosteroid (µg/day) 817 328

________________________________________________________________BMI: body mass index. *For baseline characteristics regarding lung function, see table 2.

Effects of corticosteroid withdrawal on inflammatory parameters

Sputum eosinophils (expressed both as absolute and percentage cell count)increased significantly after two-month discontinuation of ICS. No significantchanges were found in sputum neutrophils, lymphocytes, or macrophages in thisperiod, neither expressed as absolute counts or percentage of total cell count(table 3). Although sputum ECP protein levels did not change significantly afterstopping of ICS, the ECP level per sputum eosinophil decreased significantly(table 3). There was no significant change in sputum MPO levels or MPO perneutrophil. Sputum LTB4 levels increased significantly after 2-month ICSdiscontinuation (table 2). In sputum, mRNA levels of RANTES, IL-13, TGF- ,and TNF-a increased significantly after 2-month discontinuation of ICS, whereasmRNA levels of IL-5, IL-10, IL-12a, IL-12b, IFN- , and HO-1 did not changesignificantly (table 2).

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Table 2: Changes in inflammatory parameters two months after stopping inhaled corticosteroids

Visit 1 Visit 2median interquartile

rangemedian interquartile

rangep-value

Sputum cellsWeight of sputum (gram) 4.9 2.9-7.8 5.2 2.9-11.2 0.625Viability (%) 86.4 79.0-92.7 90.4 83.7-92.6 0.053Total cell count (x106/ml) 6.44 2.61-15.44 7.99 2.44-19.24 0.498Eosinophils (%) 1.5 0.5-3.2 2.3 0.8-4.7 0.003Neutrophils (%) 72.8 65.6-82.1 70.7 63.7-80.1 0.835Lymphocytes (%) 0.7 0.2-1.5 0.7 0.2-1.3 0.750Macrophages (%) 20.0 13.6-29.7 21.1 14.8-27.3 0.937

Sputum cytokinesECP (µg/l) 139.0 55.1-414.8 125.0 53.5-419.5 0.458ECP/eosinophils (µg/l /106 cells) 1509 616-3941 706 339-1745 0.003MPO (µg/ml) 12.3 6.4-37.6 11.4 5.5-52.2 0.642MPO/neutrophils(ng/ml /106 cells) 3013 1594-8172 2922 1703-6523 0.282LTB4 (ng/ml) 0.58 0.34-1.24 0.41 0.25-0.65 <0.0001

SerumCRP (mg/l) 3.3 1.9-8.3 3.3 2.2-7.7 0.848sICAM (ng/ml) 113.5 95.4-152.1 113.3 98.3-142.5 0.887

Sputum mRNA. Ct-values. n=36 completed pairsRANTES 28.9 28.6-29.6 28.6 27.9-29.4 0.008IL-5 41.1 37.3-42.9 41.3 35.9-42.6 0.388IL-10 29.9 29.3-30.6 30.1 29.1-30.9 0.480IL-12a 42.1 38.1-42.9 41.3 36.1-42.5 0.136IL-12b 41.0 37.0-42.8 41.8 37.8-42.7 0.637IL-13 41.1 36.7-42.7 36.0 34.4-42.3 0.003IFN-? 34.2 32.9-35.5 33.5 32.6-34.7 0.060TGF-ß 25.8 24.7-26.3 25.2 24.6-25.7 0.017TNF-a 26.5 26.1-27.2 26.3 25.7-26.8 0.015Haemoxygenase-1 25.5 25.1-26.1 25.5 25.0-25.9 0.418

UrineDesmosine (mM/mmol creatinin/l) 15.0 8.2-27.8 19.2 10.6-29.6 0.032

_______________________________________________________________________________ECP: eosinophilic cationic protein, MPO: myeloperoxidase, LTB4:leukotriene-B4, CRP:C-reactiveprotein, sICAM: soluble intercellular adhesion molecule, RANTES: regulated on activation, normalT-cell expressed and secreted, IL: interleukin, IFN-?: interferon-gamma, TNF-a: tumor necrosisfactor-alpha, TGF-ß: transforming growth factor-beta, HO-1: haemoxygenase-1, Ct-values: cyclethreshold values, which is the number of amplification cycles to reach detectable mRNA amount,normalised for ß2-microglobulin expression. Lower Ct-values correspond with higher mRNAexpression.

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Effects of corticosteroid withdrawal on lung function

The median (interquartile range) loss in FEV1 was 150 (80-250) ml after2-month ICS discontinuation or 4.9 (2.5 -9.3) FEV1%pred (both p<0.001).Additionally forced vital capacity (FVC), FEV1/IVC, and specific airwaysconductance (sGAW) decreased significantly (table 3).

Intra-thoracic gas volume (ITGV) expressed as percentage of total lung capacity(TLC) deteriorated significantly after 2-month ICS discontinuation (p=0.042).Trends towards detoriations occurred for residual volume (RV) expressed aspercentage of TLC (p=0.055) and inspiratory capacity (TLC minus ITGV,p=0.066) (table 2).

Table 3: Post bronchodilator lung function before and two months after stopping inhaledcorticosteroids

n=56 Visit 1 Visit 2Median Interquartile

rangeMedian Interquartile

rangep-value

FEV1 (L) 1.84 1.44-2.24 1.63 1.30-2.03 <0.0001FEV1 (%pred.) 64.3 53.0-75.4 55.5 46.9-65.8 <0.0001FVC (%pred.) 100.0 90.5-105.9 95.8 88.3-104.6 0.009IVC (%pred.) 104.2 94.1-115.5 101.7 93.6-110.1 0.079FEV1/IVC (%) 0.48 0.38-0.56 0.42 0.36-0.51 <0.0001

sGAW (kPa-1.s-1) 0.68 0.46-1.01 0.62 0.43-0.87 0.016ITGV/TLC (%pred) 108 101-116 109 102-119 0.042RV/TLC (% pred) 116 103-128 118 111-126 0.055IC (%pred) 94.3 83.8-104.9 92.4 82.4-104.5 0.066

_______________________________________________________________________________%pred: percentage of predicted value, FEV1: forced expiratory volume in one second, FVC: forcedvital capacity, IVC, slow inspiratory vital capacity, sGAW: specific airway conductance, TLC: totallung capacity, ITGV: intra-thoracal gas volume, RV: residual volume, IC: inspiratory capacity.

Effects of corticosteroid withdrawal on serum and urine parameters

Serum CRP and soluble ICAM levels did not change significantly between thetwo visits (table 3). The urinary desmosine concentration corrected for urinarycreatinin increased significantly from visit 1 to visit 2 (p= 0.03, table 3).

Effects of corticosteroid withdrawal on health status

Total CRQ and total CCQ score did not change significantly. None of thechanges in the subdomains of the questionnaires reached the level of clinicalsignificance either.

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Correlations between change in eosinophils and change in lung function

After 2-month ICS discontinuation, the increase in percentage sputumeosinophils significantly correlated with both a decrease in FEV1 (%pred) (rho =- 0.31, p= 0.020) and FEV1/IVC (rho= - 0.47, p<0.0001).

Correlations between change in eosinophils and change in eosinophilchemoattractants

The increase in eosinophil number was associated with an increase of IFN-mRNA (rho=-0.37, p=0.03). No significant associations were found betweenchanges in eosinophil numbers and mRNA expression of cytokines known toattract eosinophils like RANTES, IL-5, and IL-13, as well as LTB4 in sputumsupernatant.

Discussion

This study shows that withdrawal of inhaled corticosteroids in patients with mildto moderate COPD who remain clinically stable causes a significant increase inspecifically eosinophilic airway inflammation, which is associated withdeterioration of lung function.

The documentation of an increase in airway inflammation within two monthsafter discontinuation of inhaled corticosteroids in stable COPD patients is new.In particular mRNA for RANTES, IL-13, TGF-ß, and TNF-a and mostprominently sputum eosinophil numbers increased. The eosinophil has notalways been regarded as an important cell in the pathology of COPD. However,several studies have reported data that indicate a more prominent role ofeosinophils in COPD than initially thought. Exacerbations of chronic bronchitisare associated with a 30-fold increase in airway wall eosinophil numbers 14.Furthermore, sputum eosinophils can be present in stable COPD and highervalues correlate with more severe airway obstruction 15. Higher sputumeosinophil numbers correlate also with better improvement in lung function aftersystemic corticosteroids 16-18, inhaled corticosteroids 19-21, and even afterinhaled bronchodilator treatment 22. Furthermore, sputum eosinophil numbersare positively associated with improvement in health status after a short courseof oral corticosteroids in patients with stable COPD 18. A recent study of Barnesand colleagues showed a decrease amongst others in airway wall eosinophiliaafter starting ICS in combination with a long-acting beta-2-agonist for 13 weeksin COPD 23. Interestingly we have shown the intuitive reverse, i.e. an increase insputum eosinophils after ICS discontinuation in patients with stable COPD.

The increase in sputum eosinophils was accompanied by an increase in sputumRANTES and IL-13, both eosinophil chemoattractants. This would suggest acausal relationship as suggested in an earlier cross-sectional study24 . However,we could not find a significant correlation between the change in one of these

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eosinophil chemoattractants and the change in eosinophils. This underlines thecomplex interaction between eosinophils and the chemoattractants by whicheosinophils are recruited into the lungs 25;26. Taken together, these datasuggest a significant role of eosinophils in COPD, which can at least partially bemodified by (inhaled) corticosteroids.

Apart from the increase in eosinophils, our study showed no increases in othersputum cells after 2-month discontinuation of inhaled corticosteroids. In the past,a small study reported a non-significant increase in sputum eosinophils without achange in other sputum cells after 6 weeks ICS discontinuation in patients withirreversible airway obstruction 27. More data are available on the effects ofstarting ICS on airway inflammation. A meta-analysis of six studies using inhaledcorticosteroids for 1-12 weeks in COPD reported a reduction in sputum total cell,neutrophil, and lymphocyte counts. Studies with relatively low cumulative dose(<60 mg) or short duration of therapy (<6 weeks) did not demonstrate favourableeffects of inhaled corticosteroids on these sputum indices 21. More recently, arandomized controlled trial showed anti-inflammatory effects ofsalmeterol/fluticasone on airway inflammation compared with placebo 23. Threemonths treatment reduced the absolute number of eosinophils and percentageof neutrophils in sputum. We did not find changes in neutrophils, lymphocytes,and macrophages after 2-month discontinuation of ICS, which had been usedfor many years by most of our patients. One could speculate that a period of twomonths would be too short to find changes in the non-eosinophilic cell-types inthese patients. However, the eosinophil that is relatively steroid-sensitive provedto be a good marker of increased airway inflammation and we were able todetect this already within 2 months after corticosteroid withdrawal in stableCOPD patients.

We also found a rise in sputum TGF- , and IFN- mRNA as well as LTB4protein levels. TGF-ß is a potent mediator of fibrosis by the SMAD pathway andis produced mainly by airway epithelial cells but also by eosinophils 28. The risein TGF-ß could therefore be the result of the increased eosinophil numbers. Theincreased TGF-ß levels could have an effect on airway remodelling in COPD inthe long term. However, a negative effect of inhaled corticosteroid withdrawal onairway remodelling in COPD is not in line with the lack of effect of maintenanceICS therapy on annual lung function decline in COPD, which might reflectongoing airway remodelling. The higher sputum levels of IFN- after ICSdiscontinuation might indicate (CD8+) T-cell activation, as these cells are themain source of IFN- . In our study, LTB4 in sputum, a potent chemoattractant forneutrophils 29;30 also increased after ICS discontinuation and correlated withsputum neutrophil numbers (rho=0.28, p=0.047). The significant increases ofseveral pro-inflammatory mediators might indicate an increase in airwayinflammation after ICS discontinuation that is not yet sufficiently large to bereflected in sputum neutrophil numbers.

Discontinuation of inhaled corticosteroids resulted in an increase in airwayobstruction in the present study. This finding complements previous results fromformer corticosteroid-discontinuation studies 5;6;27. The significant increase in

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hyperinflation as assessed by ITGV/TLC after ICS discontinuation, with a similartrend for RV%TLC, is a new finding. Interestingly, the increase of hyperinflationin our study was accompanied by an increase in dyspnea, determined by CRQhealth status questionnaire (data not shown). Another study has shown thereverse, i.e. introduction of inhaled corticosteroids improved both dyspnea andhyperinflation 31. Other studies have shown that inhaled corticosteroids areeffective in improving dyspnea 32-34, yet did not investigate hyperinflation. Wepostulate that the positive effects of ICS on dyspnea may result from reductionin hyperinflation due to inhaled corticosteroid treatment.

Two months discontinuation of ICS did not worsen health status in our study, asmeasured by total CCQ or CRQ. This is more or less an expected finding givenour study design. Patients were asked to contact their clinicians when theyexperienced complaints. Nine of them did so when experiencing anexacerbation and these patients were excluded from the current analysis. Theremaining 56 patients judged themselves as stable after ICS discontinuation,which was confirmed by the outcome of the health status questionnaires.

Our study has limitations. It is not a randomized, double blind, controlled trial,the participant number is small, and the investigated period is only two months.However, the worsening of lung function after ICS discontinuation is very similarto that seen in two previous randomized controlled trials 5;6. We provide newand interesting data in that our results show that an inflammatory processoccurs in the airways of patients with stable COPD after ICS withdrawal.Additionally the statistical power of our study was sufficient to show a significantdeterioration in several parameters of inflammation among which sputumeosinophils and a significant association between an increase in sputumeosinophil numbers and increase in airway obstruction. We therefore believethat the worsening of lung function in the two previous studies and ours isactually related to the increase in airway inflammation after ICS discontinuation.There was a bias in the data available for analysis, because we were onlyinterested in clinically stable patients, i.e. patients had to be stable for twomonths after ICS withdrawal to be included in the study. However, thisstrengthens our findings, since we thus had a smaller chance to detectsignificant changes as we excluded patients from the analyses if theydeteriorated clinically. This group would indeed have deteriorated most withairway inflammation and lung function.

We conclude that discontinuation of inhaled corticosteroids in patients withstable, mild to moderate COPD causes an increase in sputum eosinophilicinflammation that correlates with a worsening of airway obstruction. This mayprovide a mechanistic basis for the deterioration of at least some patients withCOPD upon discontinuation of (inhaled) corticosteroids.

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Acknowledgement:The authors thank Jason Cook, AstraZeneca R&D, Charnwood, UK for the urinedesmosine analysis and Dr Judith Vonk, Department of Epidemiology, Universityof Groningen, the Netherlands for help with some of the statistics.

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11. Guyatt GH, Berman LB, Townsend M, Pugsley SO, Chambers LW. A measure of quality oflife for clinical trials in chronic lung disease. Thorax 1987;42:773-778.

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16. Fujimoto K, Kubo K, Yamamoto H, Yamaguchi S, Matsuzawa Y. Eosinophilic inflammationin the airway is related to glucocorticoid reversibility in patients with pulmonary emphysema.Chest 1999;115:697-702.

17. Pizzichini E, Pizzichini MM, Gibson P, Parameswaran K, Gleich GJ, Berman L, Dolovich J,Hargreave FE. Sputum eosinophilia predicts benefit from prednisone in smokers withchronic obstructive bronchitis. Am.J.Respir.Crit Care Med. 1998;158:1511-1517.

18. Brightling CE, Monteiro W, Ward R, Parker D, Morgan MD, Wardlaw AJ, Pavord ID. Sputumeosinophilia and short-term response to prednisolone in chronic obstructive pulmonarydisease: a randomised controlled trial. Lancet 2000;356:1480-1485.

19. Leigh R, Pizzichini MM, Morris MM, Maltais F, Hargreave FE, Pizzichini E. Stable COPD:predicting benefit from high-dose inhaled corticosteroid treatment. Eur.Respir.J.2006;27:964-971.

20. Brightling CE, McKenna S, Hargadon B, Birring S, Green R, Siva R, Berry M, Parker D,Monteiro W, Pavord ID, Bradding P. Sputum eosinophilia and the short term response toinhaled mometasone in chronic obstructive pulmonary disease. Thorax 2005;60:193-198.

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23. Barnes NC, Qiu YS, Pavord ID, Parker D, Davis PA, Zhu J, Johnson M, Thomson NC,Jeffery PK. Antiinflammatory effects of salmeterol/fluticasone propionate in chronicobstructive lung disease. Am.J.Respir.Crit Care Med. 2006;173:736-743.

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24. Zhu J, Qiu YS, Majumdar S, Gamble E, Matin D, Turato G, Fabbri LM, Barnes N, Saetta M,Jeffery PK. Exacerbations of Bronchitis: bronchial eosinophilia and gene expression forinterleukin-4, interleukin-5, and eosinophil chemoattractants. Am.J.Respir.Crit Care Med.2001;164:109-116.

25. Barnes PJ. Cytokine-directed therapies for the treatment of chronic airway diseases.Cytokine Growth Factor Rev. 2003;14:511-522.

26. Smit JJ, Lukacs NW. A closer look at chemokines and their role in asthmatic responses.Eur.J.Pharmacol. 2006;533:277-288.

27. O'Brien A, Russo-Magno P, Karki A, Hiranniramol S, Hardin M, Kaszuba M, Sherman C,Rounds S. Effects of withdrawal of inhaled steroids in men with severe irreversible airflowobstruction. Am.J.Respir.Crit Care Med. 2001;164:365-371.

28. Springer J, Scholz FR, Peiser C, Groneberg DA, Fischer A. SMAD-signaling in chronicobstructive pulmonary disease: transcriptional down-regulation of inhibitory SMAD 6 and 7by cigarette smoke. Biol.Chem. 2004;385:649-653.

29. Beeh KM, Kornmann O, Buhl R, Culpitt SV, Giembycz MA, Barnes PJ. Neutrophilchemotactic activity of sputum from patients with COPD: role of interleukin 8 andleukotriene B4. Chest 2003;123:1240-1247.

30. Basyigit I, Yildiz F, Ozkara SK, Yildirim E, Boyaci H, Ilgazli A. Addition of inhaledcorticosteroid on combined bronchodilator therapy in patients with COPD.Pulm.Pharmacol.Ther. 2005;18:422-426.

31. John M, Bosse S, Oltmanns U, Schumacher A, Witt C. Effects of inhaled HFAbeclomethasone on pulmonary function and symptoms in patients with chronic obstructivepulmonary disease. Respir.Med. 2005;99:1418-1424.

32. Effect of Inhaled Triamcinolone on the Decline in Pulmonary Function in ChronicObstructive Pulmonary Disease. N.Engl.J.Med. 2000;343:1902-1909.

33. Thompson WH, Carvalho P, Souza JP, Charan NB. Controlled trial of inhaled fluticasonepropionate in moderate to severe COPD. Lung 2002;180:191-201.

34. Nishimura K, Koyama H, Ikeda A, Tsukino M, Hajiro T, Mishima M, Izumi T. The effect ofhigh-dose inhaled beclomethasone dipropionate in patients with stable COPD. Chest1999;115:31-37.

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Chapter 5

Change in inflammation during COPD exacerbations

Erik Bathoorn1, Jeroen J.W. Liesker1, Dirkje S. Postma1, GerardH. Koëter1, Marco van der Toorn2, Sicco van der Heide2, AntoonJ.M. van Oosterhout2, Huib A. M. Kerstjens1

Groningen Research Institute for Asthma and COPD (GRIAC):1 Department of Pulmonology, and 2 Laboratory of Allergologyand Pulmonary Diseases, University Medical Center Groningen,University of Groningen, the Netherlands.

Submitted

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Abstract

Background: Inflammation increases during exacerbations of COPD, but only afew studies systematically assessed these changes. Better identification of thesechanges will increase our knowledge and potentially guide therapy, for instanceby helping with quicker distinction of bacterially induced exacerbations fromother causes.

Aim: To identify which inflammatory parameters increase during COPDexacerbations compared to stable disease, and to compare bacterial andnon-bacterial exacerbations.

Methods: In 45 COPD patients (37 male/8 female, 21 current smokers, meanage 65, FEV1 52% predicted, pack years 38) sputum was collected during astable phase and subsequently during an exacerbation.

Results: Sputum total cell counts (9.0 versus 7.9x106/ml), eosinophils (0.3versus 0.2x106/ml), neutrophils (6.1 versus 5.8x106/ml), and lymphocytes (0.07versus 0.02x106/ml) increased significantly during an exacerbation compared tostable disease. A bacterial infection was demonstrated by culture in 8 sputumsamples obtained during an exacerbation. These exacerbations had significantlyincreased sputum total cell and neutrophil counts, leukotriene-B4,myeloperoxidase, interleukin-8 and interleukin-6, and tumor necrosis factor-a(TNF-a) levels, and were also associated with more systemic inflammationcompared to exacerbations without a bacterial infection. Sputum TNF-a levelduring an exacerbation had the best test characteristics to predict a bacterialinfection.Conclusion: Sputum eosinophil, neutrophil, and lymphocyte counts increaseduring COPD exacerbations. The increase in systemic inflammation duringexacerbations seems to be limited to exacerbations caused by bacterialinfections of the lower airways. Sputum TNF-a is a candidate marker forpredicting airway bacterial infection.

This trial was registered at http://www.clinicaltrials.gov, ID: NCT00239278.

List of abbreviations:CCL-5= chemotactic cytokine Ligand-5CCQ= clinical COPD questionnaireCOPD= chronic obstructive pulmonary diseaseCRP= C-reactive protein

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Ct= cycle thresholdECP= eosinophilic cationic proteinFEV1= forced expiratory flow in one secondFVC= forced vital capacityHO-1= heme oxygenase-1IFN- = interferon-IL= interleukinIQR= interquartile rangeLTB4= leukotriene-B4MCP-1= monocyte chemoattractant protein-1MPO= myeloperoxidasemRNA= messenger ribonucleic acidTGF-ß= transforming growth factor-ßTNF-a= tumor necrosis factor-aVC= slow inspiratory vital capacity

Introduction

The frequent occurrence of exacerbations is an important feature of chronicobstructive pulmonary disease (COPD). The impact of exacerbations on apatient’s health is large since they are remarkably closely linked to quality of life,accelerated lung function loss, and to mortality (1;2). There is also a hugeimpact on society since the direct costs to the health care system associatedwith the management of acute exacerbations of COPD are enormous (3).Notwithstanding this, there is still no universally accepted definition of a COPDexacerbation though several have been proposed (4-7). All definitions ofexacerbation focus on symptoms, sometimes in combination with infectiousaetiology and/or airway obstruction, but remarkably, none of the definitions makeany reference to changes or increases in inflammation. This is especiallyrelevant since inflammation is part of the definition of COPD (6), and mostclinicians hold the general perception that exacerbations are associated withchanges in airway inflammation.

Airway inflammation during COPD exacerbations has been the focus of a fewstudies but their results are inconsistent (8-13). This inconsistency can beexplained by several factors. Firstly, it is difficult to gain information regardingairway inflammation during COPD exacerbations. Sputum induction by inhaledsaline can cause additional bronchoconstriction and analysis of spontaneouslyproduced sputum samples yields less cell viability (14), whereas more invasivetechniques such as bronchoscopy are even more difficult to perform duringexacerbations. For these reasons, studies have generally been performed insmall patient groups which may yield spurious results. Secondly, the causes ofCOPD exacerbations are heterogeneous. It is possible that well known inducingfactors such as viruses, bacteria, and air pollution lead to different inflammatorypatterns (6). Additionally, the specific focus of a study may well bias theselection of patients, for instance in the case of studies assessing the efficacy ofantibiotics. Thirdly, the use of medication by patients with COPD can influencethe inflammatory pattern, as is e.g. known with inhaled corticosteroids (15).

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Besides increased inflammation in the airways and the lungs duringexacerbations of COPD, an increase in systemic inflammation may occur(16;17). The cause of this altered systemic inflammation is not clear yet.However, a greater systemic inflammation has been related to the presence ofpotential pathogenic micro-organisms and the degree of cellular inflammation inthe lower airways in some studies (18;19).

Next to the limited knowledge on inflammation during an exacerbation, there isalso limited understanding of the causes of exacerbations, and, linked to this ofthe optimal treatment in relation to the cause. This has resulted in largeinternational differences in the prescription levels of most notably antibiotics,given the lack of sufficient guidance to prescribe antibiotics or none. This ispartially due to the fact that results of sputum cultures have quite a delay beforetest results are known. Thus a quicker detection of bacterial infections duringCOPD exacerbations should also lead to improvements in clinical care by aidingin the decision whether to start antibiotics. A few markers have been proposed(e.g. CRP, and procalcitonin) (20;21), but none have good test characteristics orhave gained wide acceptance so far.

The aim of the current study is to identify which inflammatory parameters areincreased in induced sputum and in blood during COPD exacerbationscompared to a stable phase of the disease, and specifically to assess whichparameters change during a bacterial exacerbation. Some of the present resultshave been described in the form of an abstract (22).

Methods

Patients with a diagnosis of COPD were included if age > 40 years,postbronchodilator forced expiratory flow in the first second (FEV1) < 85%predicted and > 0.7 litres, and postbronchodilator FEV1/slow inspiratory vitalcapacity (IVC) < predicted normal (<88% predicted in men and <89% predictedin women). Patients were not allowed to use oral corticosteroids, long-actinganticholinergics, beta-blockers, or long term oxygen therapy, have a history ofasthma, or a significant other disease that could interfere with results of thestudy. The local medical ethics committee approved the study. A writteninformed consent was obtained from all patients prior to the study.

Study design (see fig 1)In case inhaled corticosteroids (ICS) were being used at inclusion, they werediscontinued. Thereafter subjects had to be stable for 2 months. At the secondvisit, 2 months later, spirometry was performed followed by sputum induction;these measures were used as baseline, stable phase values in the study. Fromthe second visit until the end of the randomized treatment period, all long-actingbeta2-sympaticomimetics were also withdrawn. Patients were asked to contactthe research doctor, who was available around the clock, to report anydeterioration in symptoms for which they would normally contact their primary

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care physician or pulmonologist. An exacerbation was defined as a history ofincreased breathlessness and at least two of the following symptoms for 24hours or more: increased cough frequency or severity, sputum volume orpurulence, and wheeze. When presenting with an exacerbation, patients wererandomized provided postbronchodilator FEV1 was < 70% of predicted andPaO2 > 8.0 kPa. After this, a randomised controlled trial followed (data notshown).

Figure 1: Data from visit 2 and visit 3 have been used in the analyses.RCT: randomized controlled trial

MeasurementsAt each visit, sputum induction, blood sampling, and lung functionmeasurements were performed. FEV1 and IVC were measured according to theguidelines of the European Respiratory Society (23).Health status was measured by the Clinical COPD Questionnaire (24).

Sputum induction and processingSputum was induced by standard methods with modifications according toPizzichini (25). Whole sputum samples were processed within 120 minutes asdescribed previously (26). Cytospins were prepared and cell-free supernatantwas collected and stored in aliquots at -80 C pending analyses of solublemediators.Differential cell counts were counted on May Grünwald Giemsa stainedcytospins in a blinded fashion (27). Cell counts were expressed as percentage

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of non-squamous cells. A sputum sample was considered inadequate when thepercentage squamous cells was > 80%.The following soluble mediators were measured in sputum supernatant byELISA: leukotriene-B4 (LTB4, Amersham Biosciences, UK), eosinophilic cationicprotein (ECP, Pharmacia, Uppsala, Sweden), myeloperoxidase (MPO, inhouse), as well as albumin. Sputum interleukin-6 (IL-6), IL-8, tumor necrosisfactor-a (TNF-a), monocyte chemoattractant protein-1 (MCP-1), and both serumIL-6 and TNF-a were measured by xMAP technology (Luminex B.V., Oosterhout,the Netherlands), using multiplex immunoassay kits obtained from Linco, StCharles, USA.

Bacterial culturingSpontaneous sputum samples were cultured. A bacterial cause of exacerbationwas defined by the following features: the cultured micro-organisms arepotentially pathogenic, the growth density in the culture is high (semiquantitative), and the number of the leukocytes in the Gram-stained preparationof the sputum sample is >15 per high power field (100x10).

Sputum cytokine mRNA expressionMessenger ribonucleic acid (mRNA) was harvested from 1 million viablenon-squamous sputum cells. RNA was isolated using a Qiagen RNeasy mini kit(Venlo, The Netherlands) and cDNA was synthesized as described (28).Expression of cytokine mRNA was analysed by quantitative real-time PCR,using the ABI 7900 HT system (Applied Biosystems, Nieuwekerk a/d IJssel, TheNetherlands). The gene expression assays for haem-oxygenase-1 (HO-1),TNF-a, chemotactic cytokine ligand5 (CCL5), IL-5, IL-10, IL-12, IL-13,transforming growth factor- (TGF- ), interferon- (IFN- ), and

-2-microglobulin, were obtained from Applied Biosystems (Nieuwekerk a/dIJssel, Netherlands). Cytokine gene expression was normalised to theexpression of -2-microglobulin. The mRNA quantification is expressed inthreshold cycle values (Ct-values), which is the number of amplification cycles toreach a detectable mRNA amount. Thus lower Ct-values correspond with highermRNA expression.

Blood analysesBlood differential counts were analysed by flow cytometry (Coulter-STKS,Beckman Coulter, Miami, USA). Serum C-Reactive Protein (CRP) and albuminwere measured by nephelometry (Dade Behring, Leusden, the Netherlands).

Statistical analysisData are expressed as medians and inter quartile ranges (IQR). Non-normallydistributed parameters were normalised by log10 transformation. Stable phaselevels of inflammatory parameters were compared to exacerbation levels usingpaired sample t-tests, or Wilcoxon log rank tests. Exacerbations with andwithout a bacterial infection were compared with respect to both the

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cross-sectional values at exacerbation and the differences in the % change ofbiomarkers from baseline to exacerbation (t-tests or Mann-Witney U-test). UsingReceiver Operating Curves, cut-off points for the biomarkers were adjusted untilthe highest area under the curve was reached. Data were analysed using SPSSversion 12.0.2.

Results

We included 114 patients in this study. The 45 patients reporting anexacerbation within the study period were analysed. The median time toexacerbation from the stable visit 2 months after ICS withdrawal was 81 days.The baseline characteristics of these patients are presented in table 1. Duringthe exacerbation, patients had a significantly and clinically relevant poorer healthstatus as measured by higher CCQ scores compared to the stable phase(median value 2.5 versus 1.7 respectively, p<0.01). The minimal clinicallyimportant difference of the CCQ is 0.4 (29).

Table 1. Patient characteristics

n=45Male/female 37/8 Age, years* 65 (58-71)Smoked, packyears* 38 (26-49)Smoking status, current/ex 21/24 Body mass index, kg/m2* 25 (24-28)FEV1, % pred* 61 (48-73)FEV1/IVC, % 44 (38-53)Reversibility, % of pred* 8.9 (5.3-11.0)CCQ-score* 1.7 (1.3-2.1)

_______________________________________________________________________________*median (interquartile range)n= numberkg= kilogramsm= meterspred= predictedCCQ= Clinical COPD Questionnaire

Inflammatory indices

Sputum samples were adequate (<80% squamous cells) in both the stablephase, after ICS withdrawal, and during exacerbation in 41 out of 45 patients.The cellular differences between the stable phase (2 months after ICSwithdrawal) and during the exacerbation are presented in table 2. Sputum totalcell, eosinophil, neutrophil, and lymphocyte counts were significantly increasedat exacerbation versus stable disease.

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Analyses of mRNA levels in sputum showed a lower IL-12a expression atexacerbation compared to stable state values (median ct-values 41.5 versus37.7 respectively; p=0.051). No differences were found in sputum mRNA levelsof HO-1, TNF-a, IL-5, CCL5, IL-10, IL-12b, IL-13, TGF-ß, and INF-gamma (datanot shown). The sputum protein levels of ECP, MCP-1, and LTB-4, weresignificantly increased at exacerbation compared to stable state and levels ofMPO and IL-8 tended to be increased (table 3).

Systemic inflammation during COPD exacerbations was increased compared tothe stable phase, i.e. blood leukocyte and neutrophil counts were significantlyincreased (table 2), as were serum protein levels of IL-6 (table 3). There was atrend towards an increase in CRP (p= 0.07).

Table 2. Cellular inflammatory parameters_______________________________________________________________________________ Stable Exacerbation

Sputum:Total cells x106/mL 7.9 (2.1-19.2) 9.0 (4.0-29.8) *Eosinophil % 2.7 (0.8-5.8) 2.8 (0.8-4.8)Eosinophil x106/mL 0.2 (0.1-0.4) 0.3 (0.1-0.7) *Neutrophils % 71.7 (65.3-80.0) 72.0 (62.1-86.1)Neutrophils x106/mL 5.8 (1.5-13.4) 6.1 (2.1-24.1) *Macrophages % 20.7 (14.5-26.8) 17.7 (10.1-24.9)Macrophages x106/mL 1.4 (0.5-3.7) 2.1 (0.6-4.1)Lymphocytes % 0.3 (0.0-1.3) 0.8 (0.3-1.3) *Lymphocytes x106/mL 0.02 (0.00-0.10) 0.07 (0.03-0.20) *

Blood:Leukocytes x109/mL 6.6 (6.0-8.2) 7.5 (5.9-9.1) *Eosinophil % 3.2 (2.2-5.8) 2.9 (1.9-5.8)Eosinophil x109/mL 0.2 (0.2-0.3) 0.2 (0.1-0.4)Neutrophils % 61.0 (55.4-65.9) 63.0 (57.2-68.2)Neutrophils x109/mL 4.2 (3.5-4.9) 4.6 (3.4-6.1) *Monocytes % 10.1 (8.3-11.0) 8.8 (7.9-10.8)Monocytes x109/mL 0.6 (0.5-0.8) 0.7 (0.6-0.8)Lymphocytes % 24.4 (20.2-28.8) 22.9 (20.4-27.1)Lymphocytes x109/mL 1.6 (1.4-2.0) 1.7 (1.4-2.0)___________________________________________________________________________41 of 45 patients had an adequate sample for sputum cell differential analysis at both stable phase and exacerbation. Median (interquartile range). *: significant difference (p<0.05)

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Table 3. Inflammatory biomarkers_______________________________________________________________________________ Stable Exacerbation p-valueSputum:LTB-4 ng/mL 0.4 (0.2-0.7) 0.5 (0.3-1.1) 0.03TNF-a pg/mL 1.6 (1.6-8.7) 4.6 (1.6-31.5) 0.37IL-6 pg/mL 287 (183-637) 361 (164-783) 0.24IL-8 µg/mL 1.5 (0.9-4.4) 2.0 (1.2-6.3) 0.07MCP-1 pg/mL 119 (63-560) 210 (137-385) 0.01MPO µg/mL 9.2 (5.3-31.0) 15.1 (8.4-56.0) 0.08ECP µg/L 85.4 (49.6-319.5) 187.0(81.3-295.0) 0.03

Serum:IL-6 pg/mL 0.4 (0.1-4.0) 0.6 (0.1-9.9) 0.03TNF-a pg/mL 4.9 (3.3-6.8) 4.5 (2.6-6.9) 0.90sICAM ng/mL 109 (91-129) 118 (105-132) 0.13CRP mg/L 3.4 (1.8-8.2) 3.6 (1.5-17.1) 0.07

Sputum/Serum:Albumin ratio 1.7 (0.9-3.5) 2.0 (1.1-3.3) 0.03___________________________________________________________________________

Median (interquartile range). Analysed for differences with paired t-tests.LTB4: leukotriene-B4, TNF-a: tumor necrosis factor-a, IL-6: interleukin-6, IL-8: interleukin-8,MCP-1: monocyte chemoattractant protein-1 MPO: myeloperoxidase, ECP: eosinophiliccationic protein, sICAM: soluble intercellular adhesion molecule CRP: C-reactive protein

Bacterial culturesEight sputum samples for bacterial culture during exacerbation of COPD weremissing: 5 patients produced too small a volume of spontaneous sputum, and 3samples were missing due to logistical problems. Eight of the remaining 37sputum samples were indicative of a bacterial infection: 5 with Haemophilusinfluenzae, 2 with Moraxella catarrhalis, and 1 with Streptococcus pneumoniae.In stable phase, 10 patients produced too small a volume of spontaneoussputum and 1 was missing due to logistical problems. Two of the remaining 34samples were indicative of a bacterial infection (Haemophilus influenzae andcombination of Haemophilus influenzae and Moraxella catarrhalis).

Differences between bacterial and non-bacterial exacerbationsTable 4 shows the differences between bacterial and non-bacterialexacerbations both cross-sectionally at the exacerbation visit, and as a changefrom baseline with stable disease to the exacerbation in order to correct forstable phase values.Bacterial exacerbations were accompanied by higher values for sputum total celland neutrophil counts, LTB4, MPO, IL-8, and TNF-a level than non bacterialexacerbations. In serum, bacterial exacerbations were associated with highertotal leukocytes and neutrophil counts. Additionally, when analysed as the %change from baseline to exacerbation, serum CRP and IL-6 levels showed asignificantly larger increase when a bacterial exacerbation occurred compared toexacerbations without a bacterial infection.The predictive values of the level of blood CRP, blood leukocytes, sputumTNF-a and sputum MPO for a bacterial cause of the exacerbation are presented

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in Receiver Operating Curves (figure 2). Sputum TNF-a had the best testcharacteristics.

Table 4: Differences between bacterial and non-bacterial exacerbations._______________________________________________________________________________

Cross-sectional values atexacerbation

% Change from stable phase toexacerbation

Bacterialexacerbation

Non-bacterialexacerbation

Bacterialexacerbation

Non-bacterialexacerbation

Sputum

total cell x106 /mL †† 51.1 (21.2-66.6)*

7.60 (2.94-22.9) 269 (44-3183)* -5 (-36-95)

eosinophil x106 /mL †† 0.49 (0.22-1.30) 0.29 (0.06-0.79) 134 (-22-1460) 51 (-34-174)

neutrophil x106 /mL †† 47.4 (13.2-60.2)*

4.85 (1.91-17.2) 203 (35-760)* 4 (-39-87)

lymphocyte x106 /mL ‡† 0.22 (0.05-0.56) 0.07 (0.03-0.21) 949 (33-3815) 114 (-31-482)

ECP µg/mL †† 247 (139-1001) 187 (96.9-279) 137 (9-222) 50 (-20-183)

LTB4 ng/mL ‡‡ 1.43 (0.71-2.01)*

0.43 (0.34-0.59) 164 (50-347)* 0 (-26-54)

MPO µg/mL †† 57.7 (30.4-89.9)*

12.6 (6.29-34.6) 349 (-2-955) 7 ( -34-102)

IL-6 pg/mL †† 680 (267-1251) 325 (153-733) 116 (52-610)* -16 (-47-77)

IL-8 µg/mL † † 7.78 (3.52-9.90)*

1.74 (0.92-4.47) 149 (0-444) 6 (-23-72)

TNF-a pg/mL ‡‡ 56.8 (43.3-69.7)*

3.43 (1.60-7.73) 1580 (91-583) ‡ 0 (-17-110)

MCP-1 pg/mL †† 283 (177-799) 194 (131-360) 141 (11-376) 41 (-32-171)

Sputum/Serum albumin‡‡ 3.06 (2.44-3.65) 1.99 (1.14-3.11) 51 (15-85) 35 (-21-100)

Blood/serum

leukocytes x106 /ml †† 8.95 (7.95-10.3)*

6.60 (5.70-8.15) 24 (1-53) ‡ 2 (-5-18)

neutrophil x106 /ml †† 6.47 (4.87-7.62)*

3.84 (3.34-5.63) 23 (5-69) 4 (-14-25)

sICAM µg/mL †‡ 11.2 (10.3-13.9) 12.1 (10.5-13.3) 11 (-5-26) 4 (-3-13)

CRP mg/L ‡‡ 9.08 (4.56-26.2) 2.6 (1.4-15.3) 143 (39-740) ‡ 1 (-42-87)

IL-6 pg/mL ‡‡ 4.86 (1.22-10.0) 0.40 (0.10-10.7) 670 (119-2225)‡ 0 (-2-126)

TNF-a pg/mL ‡† 4.50 (2.33-7.36) 4.60 (2.61-7.00) -7 (-23 – 21) -11 (-21-13)

_______________________________________________________________________________Data presented as median (IQR). †: Tested for differences by T-test ‡: Tested for differences byMann-Witney-U-test. *: p<0.05 for the difference in cross-sectional values between bacterial andnon bacterial exacerbations; ‡:p<0.05 for the difference in percentage change from stable toexacerbation values between bacterial and non-bacterial exacerbations.LTB4: leukotriene-B4, TNF-a: tumor necrosis factor –a, IL-6: interleukin-6, IL-8: interleukin-8,MCP-1: monocyte chemo-attractant protein-1 MPO: myeloperoxidase, ECP: eosinophilic cationicprotein, CRP: C-reactive protein

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Figure 2: Test characteristics of inflammatory markers for a bacterial cause of exacerbations

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Discussion

This study is the first to assess the change in inflammation from a stable phaseof COPD to a COPD exacerbation in patients who did not use inhaledcorticosteroids for at least 2 months. Sputum neutrophil, lymphocyte, andeosinophil numbers increased during an exacerbation. Furthermore, systemicinflammation increased during exacerbations as assessed by blood totalleukocyte and neutrophil counts, and serum CRP and IL-6. These biomarkerswere particularly increased in case of bacterial exacerbations. The level ofsputum TNF-a during an exacerbation was the best predictor of a bacterialairway infection.

As expected, there was an increase in sputum neutrophils during COPDexacerbations (10;13;30). The increase in sputum neutrophil numbers wasaccompanied by an increase in sputum levels of LTB4 and IL8, both attractantsof neutrophils, and the neutrophil degranulation product MPO. These changesconfirm an earlier study of Gompertz et al (31), reporting a decrease in airwayinflammation from the start of a COPD exacerbation to its resolution. Theseauthors also reported a change in microvascular leakage as determined by thesputum/serum albumin ratio, which we confirm in our prospective study.

Furthermore, we found an increase of sputum lymphocytes during exacerbations(10;13;30). Lymphocytes play an important role in the pathogenesis of COPD.They are related to its development and progression. Lams et al found thatsmokers who develop COPD have increased CD8+ T-cells in large airwayscompared to asymptomatic smokers (16). Furthermore, there is an increase inCD4+ cells in patients with COPD, particularly as the disease progresses (32).This increase in lymphocytes has been suggested to be caused by chronicimmune stimulation due to infectious pathogens (33). Not only T-cells, but alsoB-cells are increased in stable COPD (34-36). Viral airway infections maycontribute in this respect (37;38), but an autoimmune origin has also beensuggested (34;39;40). In the present study we were not able to identify viruses ininduced sputum samples. Whether part of the increase in lymphocytes in ourstudy is of B-cell origin was not evaluated.

This is at least the forth report documenting an increase in eosinophil countsduring exacerbations (12;30;41). Next to the increase in eosinophils, we alsofound increased sputum ECP. ECP has been associated with tissue damageand tissue remodelling in in vitro studies (42). This might thus contribute to theobserved association between exacerbation frequency and excess decline inlung function (1). However, not all prior studies have found increases ineosinophils during COPD exacerbations (9). This difference between our andother’s observation of an increase in eosinophils may be simply explained by asuppressive effect of the used steroids on sputum eosinophils in these studies(15).

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Next to increased sputum inflammation as proxy of airways inflammation, wealso found increased systemic inflammation as demonstrated by increasednumber of blood total leukocytes, neutrophils, IL-6, and a trend in CRP duringexacerbations. This confirms an earlier report of increased systemicinflammation (increased serum IL-6 and CRP) during COPD exacerbations(19;43). In 22% of our sputum samples the exacerbation was associated with abacterial infection. These exacerbations showed a higher increase of total celland neutrophil counts, IL-6, TNF-a levels in sputum, and leukocyte andneutrophil counts, CRP, and IL-6 levels in blood. This relationship of highersputum and systemic inflammation in the presence of a bacterial pathogen insputum has been reported in earlier studies. However, these studies did notinvestigate whether these high levels were already present in stable state(13;19) or, as we now show, indeed reflected an increase that occurs with abacterial infection. Our data shows that some of the biomarkers that areincreased in an exacerbation actually only change if the specific exacerbation isinduced by a bacterial infection.

Serum CRP has been investigated as a marker to identify exacerbations in onestudy (20). The predictive value of serum CRP alone was limited in that study,and use of the combination of the CRP level with a symptom of exacerbationwas proposed to improve the predictive value of CRP. Our study shows thatCRP is much more increased in bacterial exacerbations. We believe thisunderlines the limitations of CRP as a general biomarker of COPDexacerbations.

It would be of clinical use if clinicians would have biomarkers with cut-off pointsto differentiate which patient has a bacterial airway infection. If one would knowfrom a rapidly available biomarker whether there is a high chance of a bacterialinfection, this would improve the efficiency of use of antibiotic treatment. Ourstudy population had mild to moderate COPD and is not large enough to providefirm data on such cut-off points, and we investigated exacerbations that did notrequire hospitalisation. Nevertheless, we do present the ROC-curves to offerreference and directions for future assessment of firm cut-off points. From ourpreliminary analyses, sputum TNF-a seems to be a good candidate biomarker infuture studies.

In summary, this study showed an increase in neutrophilic, eosinophilic, andlymphocytic airway inflammation from a stable phase of disease to anexacerbation in COPD patients withdrawn from inhaled corticosteroids. Inaddition, systemic airway inflammation increased during exacerbations and ofinterest, this was limited to exacerbations with bacterial infections. Some of thebiomarkers, specifically sputum LTB4, MPO, IL-6, TNF-a, and serum CRP andIL-6, which are commonly associated with exacerbations, are increased duringbacterial exacerbations, but little or not increased at all during non-bacterialexacerbations. Though our group of patients was too small to confer firmconclusion, the data lend support to further investigate whether theseinflammatory parameters, and specifically TNF-a provides a useful tool foridentification of a bacterial infection in COPD.

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Acknowledgements

The authors thank Ibolya Sloots, Brigitte Dijkhuizen, Koos van de Belt, andJanneke Heimweg for the sputum measurements, the lung function departmentfor the many lung function measurements, and Dr. N.E.L. Meessen for his helpwith the interpretation of sputum culture results.

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(39) Majo J, Ghezzo H, Cosio MG. Lymphocyte population and apoptosis in the lungs ofsmokers and their relation to emphysema. Eur Respir J 2001; 17(5):946-953.

(40) Agusti A, MacNee W, Donaldson K, Cosio M. Hypothesis: does COPD have anautoimmune component? Thorax 2003; 58(10):832-834.


(42) Zagai U, Skold CM, Trulson A, Venge P, Lundahl J. The effect of eosinophils on collagengel contraction and implications for tissue remodelling. Clin Exp Immunol 2004;135(3):427-433.

(43) Seemungal T, Harper-Owen R, Bhowmik A, Moric I, Sanderson G, Message S et al.Respiratory viruses, symptoms, and inflammatory markers in acute exacerbations andstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164(9):1618-1623.

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Chapter 6

Anti-inflammatory effects of combinedbudesonide/formoterol in COPD exacerbations.

Erik Bathoorn1, Jeroen Liesker1, Dirkje Postma1, MartinBoorsma2, Eva Bondesson3, Gerard Koëter1, Henk Kauffman4,Antoon van Oosterhout4 and Huib Kerstjens1.

Groningen Research Institute for Asthma and COPD (GRIAC),Department of Pulmonology1 and Laboratory of Pulmonology andAllergology4, University Medical Center Groningen, University ofGroningen, Groningen, the Netherlands, and AstraZeneca R&D,Zoetermeer, The Netherlands2 AstraZeneca R&D, Lund,Sweden3.

Submitted

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Abstract

Systemic corticosteroids and additional short-acting ß2-agonists are commonlyused in exacerbations of chronic obstructive pulmonary disease (COPD). In thisdouble-blind study, the combination of a high dose inhaled corticosteroid with arapid-onset long-acting ß2-agonist was evaluated in the treatment of out-patientCOPD exacerbations.The primary aim was to compare 14-day treatment effects ofbudesonide/formoterol to placebo on sputum eosinophils and, secondarily, onother indices of inflammation, forced expiratory flow in one second (FEV1),symptoms, health status, and adverse events.Forty-five patients not using steroids (37 male, 21/24 current/ex smoker, medianpackyears 38, age 65 years, FEV1 61% predicted) experiencing a COPDexacerbation were treated at home with 160µg budesonide/ 4.5µg formoterol (2inhalations q.i.d), prednisolone (30 mg daily), or placebo for 14 days.Sputum eosinophils were significantly reduced by budesonide/formoterol (-57%)compared to placebo (+24%) (p=0.01). Budesonide/formoterol reduced totalsymptom scores significantly (p=0.01) compared to placebo. The increase inFEV1 by 2 weeks of treatment with budesonide/formoterol (125 ml) was notsignificantly different from that of placebo (43 ml) (p=0.07).Budesonide/formoterol treatment did not suppress morning serum cortisolcompared to placebo (-16 %; p=0.50).In conclusion, budesonide/formoterol reduces sputum eosinophils and improvessymptoms in the treatment of out-patient COPD exacerbations.

This study has been registered at http://www.clinicaltrials.gov, ID:NCT00239278.

Keywords: budesonide, chronic obstructive pulmonary disease, exacerbation,formoterol inflammation, sputum induction.

Introduction

Exacerbations of COPD are traditionally treated with systemic corticosteroidsand short-acting bronchodilators, with or without antibiotics. The effectiveness ofsystemic steroids, though used for decades, has only recently become“evidence based” 1;2. A theoretically alternative treatment of COPDexacerbations, avoiding the use of systemic corticosteroids, is treatment withinhaled corticosteroids 3. Additionally, immediate bronchodilation can also beobtained with the long-acting ß2-agonist formoterol due to its fast onset ofaction. This is an attractive option, since patients who already use combinedinhaled corticosteroids and long-acting ß2-agonists as maintenance therapy,such as budesonide/formoterol could increase their daily dose when they areexperiencing early signs of an exacerbation, thereby possibly preventing a fullblown exacerbation. Such a treatment approach has been shown to be effective

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Anti-inflammatory effects of combined budesonide/formoterol in COPD exacerbations

in the treatment of asthma 4. Thus far, the treatment of mild to moderate COPDexacerbations with an inhaled corticosteroid and a long acting ß2-agonistcombined has not been tested in a randomised controlled trial. The combinationhas proven efficacy in preventing exacerbations of COPD 5;6.

Steroids are thought to exert their beneficial effect on exacerbations of COPDthrough their anti-inflammatory properties. Effects of systemic steroids oninflammation during exacerbations have not been reported, although somestudies document changes in stable phase COPD 7;8, effects that are alsoobserved with inhaled steroids 9-12. Effects of ß2-agonists depend largely ontheir bronchodilating properties. There are no data of anti-inflammatory effectsof ß2-agonists in COPD, but in asthma anti-inflammatory properties have beendescribed 13;14. Although inflammation assessed in sputum in stable phase ofCOPD is mainly of neutrophilic origin, several studies have indicated thatsputum eosinophils are increased during COPD exacerbations 15;16.Furthermore sputum eosinophilia predicts a better response to a short-termsteroid treatment in a stable phase of COPD 17-19. We hypothesised that theclinical response to steroids in COPD exacerbations is due to the suppressiveeffect on the eosinophilic inflammatory component and therefore designated thiscell as the primary efficacy endpoint to assess inflammatory treatment effects.

We examined whether a combination therapy with an inhaled corticosteroid andlong-acting ß2-agonist, i.e. budesonide and formoterol (B/F), as available in asingle inhaler would reduce inflammation and especially the eosinophiliccomponent occurring during exacerbations, more effectively than placebo(PLAC). We estimated that documentation of effects on clinical parameterswould require more patients and therefore pre-defined these parameters assecondary outcomes only.

Some of the results of this study have been previously reported in the form of anabstract 20;21.

Methods

Subjects

Inclusion criteria for the study were: diagnosis of COPD, age >40 years,postbronchodilator FEV1 <85% predicted but >0.7 liters, and an abnormalpostbronchodilator FEV1/slow inspiratory vital capacity (VC) (<88% predicted inmen and <89% predicted in women) 22. Patients were not allowed to use oralcorticosteroids, longacting anticholinergics, beta-blockers, or oxygen therapy, orhave a history of asthma or significant other disease that could influence theresults of the study. The study was performed in accordance with the principlesstated in the Declaration of Helsinki. The local medical ethics committeeapproved the study. Written informed consent was obtained from all patients.

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Study design

At the inclusion visit, inhaled corticosteroids were discontinued if used. After thisvisit, the subjects had to be stable during a run-in period of 2 months. After thewash-out period, spirometry was performed followed by a sputum inductionprocedure, and long-acting ß2-agonists were withdrawn. Patients contacted theresearch facility if they experienced an exacerbation, as defined according toDavies 23: a history of increased breathlessness and at least two of the followingsymptoms for =24 hours: increased cough frequency or severity, sputum volumeor purulence, and wheeze. When presenting with an exacerbation, and providedthat postbronchodilator FEV1 < 70 % predicted and PaO2 > 8.0 kPa, patientswere randomised to a):160µg budesonide/ 4.5µg formoterol (SymbicortTurbuhaler®, AstraZeneca Sweden), 2 inhalations q.i.d., b): 30 mg prednisolone(AstraZeneca) as 6 tablets of 5 mg once daily, or c): placebo tablets andinhalations, in a double dummy set-up, for 14 days. Inhalation technique waspractised until satisfactory. All patients received a standard dose of doxycyclin,and inhaled terbutaline and ipratropium bromide as needed. For the allocation torandomised treatment we used sputum eosinophils (< or = 3%), FEV1 atexacerbation (< or = 50% of predicted), smoking status (current or ex), inhaledcorticosteroid and N-acetyl-cysteine use at start of study as stratification factorsin a minimization process (see online supplement for details on randomisationprocess).

Measurements

At randomization, and at day 3, 7, and 14, induced sputum, blood, and urinewere collected, an electrocardiogram was made and heart rate, weight, bloodpressure, and lung function were measured. Patients recorded morning andevening symptoms of breathlessness, sputum, and cough daily in a diary in thelast 2 weeks of the wash-out period and during 2 weeks after randomisation 24.Patients filled in the Clinical COPD Questionnaire (CCQ) at each visit, and theClinical Respiratory Questionnaire at baseline and at the and of treatment 25.FEV1, VC, forced vital capacity (FVC), and specific airways conductance (sGaw)(Masterscreen Bodybox, Jäeger, Würzburg, Germany) were measuredaccording to guidelines of the European Respiratory Society 22.

Sputum induction and processing

Sputum was induced by standard methods 26, with modifications according toPizzichini when the FEV1 was <1.5 liters 27. Messenger Ribonucleic Acid(mRNA) processing by real-time polymerase chain reaction and other methodsare described in the online supplement.

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Statistical analysisThe primary endpoint was the change in % sputum eosinophils defined as theratio of the % eosinophils at day 14 to the % eosinophils of the randomisationvisit (day 1). Using GAUSS from Aptech systems inc. (kernel revision 6.0.48),ratios were compared in a multiplicative analysis of variances model with thevalue of the randomisation visit included as a covariate. Pairwise treatmentratios and 95% confidence intervals were compared in the model using contrast.Secondary endpoints were analysed using similar methods. Power calculationwas based on the change in eosinophils by steroid treatment in stable COPD ina previous study, demonstrating a change in eosinophils of 1.5% (see onlinesupplement).

Figure 1: Flow chart. B/F= Budesonide/ Formoterol; PRED= Prednisolone; PLAC= Placebo

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Results

Subjects

In the study 114 patients were recruited, of whom 45 patients reported anexacerbation after the run-in period and were randomised (figure 1). Thebaseline characteristics of the randomised patients are presented in table 1.Two patients were withdrawn due to treatment failure (1 in the placebo groupafter 1 day and 1 in the B/F group after 7 days of treatment) The other 43patients all completed the 2 weeks of randomised double-blind treatment.

Table 1. Patient characteristics____________________________________________________________________________

budesonide/formoterol prednisolone placebo

Number of patients 15 15 15

Sex (male/female) 10/5 13/2 14/1

Age (years) 61.4 (8.0) 64.8 (7.0) 64.6 (9.1)

Smoking status, current/ex 6/9 8/7 7/8

Packyears † 39 (28-75) 38 (30-48) 32 (19-49)

Body mass index (kg/m2) 25.7 (4.3) 25.7 (3.8) 25.1 (3.1)

FEV1 (% predicted), atenrolment

63.5 (13.0) 60.2 (14.4) 57.4 (12.7)

FEV1 (% ) at exacerbation 54.1 (14.3) 52.6 (16.0) 49.6 (13.4)

FEV1/VC at enrolment 46.8 (10.1) 43.8 (12.6) 45.5 (10.4)

FEV1/VC at exacerbation. 41 (11) 38 (10.5) 38 (8.5)

Reversibility (% predicted) 8.9 (4.6) 7.7 (6.4) 9.8 (6.5)

_______________________________________________________________________________Data as actual numbers or as mean (sd). †: median (interquartile range). One patient in theplacebo group needed open label treatment at day 2; data of the remaining 44 patients wereused for analysis. Reversibility was measured after 2 months inhaled steroid withdrawal.

Inflammatory cells

Treatment with B/F and PRED elicited a significant decrease versus placebo inthe percentage sputum eosinophils (figure 2): the mean reduction after 2 weeks

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was 57% for B/F compared to an increase with 24% for placebo (p=0.01) and areduction of 58% for PRED (p=0.007 versus placebo). No difference betweenthe two active treatments was found, nor were there any significant differencesin the other sputum cell differential counts or in the total cell counts between thetreatment groups (table 2). Blood eosinophil counts were not affected by eitherof the two active treatments. PRED treatment resulted in an increase in bloodleucocyte count (mean change 3.1x 109/L and blood neutrophils (mean change1.91 x109/L) which was significantly different from PLAC (p<0.001 and p=0.01respectively).

Table 2. Sputum cells


Total cells (x106/mL) day 1 7.9 (183) 16.5 (355) 7.0 (165)

Total cells (x106 mL) day 14 4.0 (153) 11.4 (749) 4.3 (178)

Eosinophil % day 1 2.6 (377) 2.0 (163) 2.4 (186)Eosinophil % day 14 0.9 (234)* 0.8 (200) † 3.5 (97)

Eosinophil (x106 /mL) day 1 0.21 (281) 0.32 (686) 0.21 (280)

Eosinophil (x106/mL) day 14 0.03 (847)* 0.09 (1274) 0.19 (204)

Neutrophils % day 1 66.3 (26) 80.6 (12) 67.1 (22)Neutrophils % day 14 69.9 (24) 80.5 (11) 68.1 (12)Macrophages % day 1 17.2 (63) 12.1 (39) 17.8 (87)Macrophages % day 14 18.6 (50) 13.0 (58) 21.5 (48)Lymphocytes % day 1 1.0 (91) 0.6 (86) 0.7 (138)

Lymphocytes % day 14 0.7(120) 0.8 (75) 0.4 (81)_____________________________________________________________________________Data presented as geometric mean and coefficient of variation (sd/mean x 100%).*: p<0.05 budesonide/formoterol vs placebo. †: p<0.05 prednisolone vs placebo. P-values forcomparisons of the ratio at day 14 to day 1 under budesonide/formoterol (320/9 µg 4 times daily)versus prednisolone (30 mg once daily) and placebo.

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Figure 2: Treatment effect on sputum eosinophil%. * On day 3, 7, and 14, the ratio of sputumeosinophil % of the visit under study to the eosinophil% at randomisation are presented. Thedifference in ratios from start to end of treatment are significant for budesonide/formoterol versusplacebo (p=0.01) and for prednisolone versus placebo (p=0.007). Data is expressed as geometricmeans. P-values for comparisons of these ratios at day 14 under budesonide/formoterol (320/9 µg4 times daily) versus prednisolone (30 mg once daily) and placebo.

Sputum mRNAThe Ct-values of the mRNA expression are shown in table 3. B/F treatmentresulted in a significantly larger decrease in interleukin-5 (IL-5) expressioncompared to placebo (p=0.02). PRED treatment resulted in a smaller increase inexpression of heme oxygenase-1 (HO-1) compared to B/F (p=0.02) and in asmaller increase in the expression of transforming growth factor- ß (TGF-ß)mRNA compared to PLAC (p=0.045).

Lung functionThe change in FEV1 is shown in figure 2. FEV1 improved during the treatmentperiod in all three groups. The increase in FEV1 by 2 weeks of treatment withbudesonide/formoterol (125 ml) was not significantly different from that ofplacebo (43 ml) (p=0.07). With PRED the FEV1 improved 27 ml (p=0.71 versusplacebo). There were no significant differences in the effects of B/F orprednisolone versus placebo after 2 weeks on other lung function parameters(table 4).

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Table 3. mRNA expression of sputum cells


mRNA HO-1, 0.5*106 day 1 25.23 (4.2) 26.27 (3.1) 25.48 (3.9)

mRNA HO-1, 0.5*106 day 14 24.97 (5.5) 26.02 (3.3) ‡ 24.97 (3.3)

mRNA TNF-a, 0.5*106 day 1 26.80 (4.3) 25.77 (4.8) 26.27 (3.7)

mRNA TNF-a, 0.5*106day 14

26.83 (5.4) 26.05 (4.9) 26.45 (3.7)

mRNA TGF-ß, Ct day 1 25.52 (6.1) 25.41 (3.4) 24.96 (3.3)

mRNA TGF-ß, Ct day 14 24.97 (4.0) 25.23 (3.2) † 24.35 (4.2)

mRNA INF-gamma, Ct day 1 33.02 (6.0) 34.27 (9.3) 33.67 (8.5)

mRNA INF-gamma, Ct day14

33.59 (6.8) 34.78 (9.3) 34.31 (8.3)

mRNA IL-10, Ct day 1 31.09 (5.2) 29.98 (3.8) 30.14 (2.6)

mRNA IL-10, Ct day 14 31.22 (10.5) 29.66 (4.6) 30.20 (4.2)

mRNA IL-12a, Ct day 1 37.81 (10.9) 40.43 (9.3) 39.05 (9.2)

mRNA IL-12a, Ct day 14 38.20 (11.5) 40.26 (6.7) 38.31 (11.1)

mRNA IL-12b, Ct day 1 39.27 (10.5) 36.99 (6.6) 41.85 (5.2)

mRNA IL-12b, Ct day 14 41.08 (4.6) 38.97 (8.3) 40.75 (6.4)

mRNA IL-5, Ct day 1 35.89 (11.1) 38.61 (8.6) 37.77 (14.0)

mRNA IL-5, Ct day 14 40.44 (5.9)* 37.70 (8.6) 38.69 (10.0)

mRNA IL-13, Ct day 1 35.89 (11.2) 38.02 (10.3) 37.04 (10.6)

mRNA IL-13, Ct day 14 39.68 (7.9) 38.06 (10.3) 37.50 (8.7)

mRNA CCL5, Ct day 1 29.34 (7.2) 28.85 (2.9) 28.25 (3.9)

mRNA CCL5, Ct day 14 28.69 (3.7) 29.04 (3.4) 28.50 (2.4)

_______________________________________________________________________________mRNA expression of sputum cells. The Ct-values at end of treatment minus the values at start oftreatment are presented. Data presented as geometric mean and coefficient of variation (sd/mean x100%). *: p<0.05 budesonide/formoterol vs placebo. †: p<0.05 prednisolone vs placebo. ‡: p<0.05budesonide/formoterol vs prednisolone. Lower Ct-values correspond with higher mRNAexpression. Ct-values are normalised for ß2-microglobulin expression. *: P-values for comparisonsof the ratio at day 14 to day 1 under budesonide/formoterol (320/9 µg 4 times daily) versusprednisolone (30 mg once daily) and placebo. HO-1= heme ogygenase-1

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Symptoms and health status

During both B/F and PRED treatment periods, significantly lower total symptomscores in the diaries were observed compared to the treatment period withPLAC (mean difference -1.37 and -1.03 respectively; p<0.01, p=0.048). B/Ftreatment resulted also in lower cough symptom score compared to PLAC(mean difference -0.62, p=0.015). There were trends towards lower sputumproduction scores under both B/F and PRED compared to PLAC (meandifferences –0.38 and –0.39 respectively, p=0.066 and p=0.058).The overall health status as measured by the CCQ did not differ significantlybetween B/F and PLAC (mean change 1.0 and 0.5 points, respectively: p=0.08)(figure 4). However, the improvement in overall health status with B/F wassignificantly greater compared to that with PRED (mean change 0.4 points,p=0.02). The minimal clinical important difference of the CCQ is a change of 0.4points28. The health status measured by the CRQ showed similar results (meanchange B/F 0.47 and placebo 0.004, p=0.07; mean change PRED –0.06, p=0.04versus B/F).

Figure 3: Treatment effect on FEV1* On day 3, 7, and 14, the ratio of FEV1 of the visit under study to the FEV1 at randomisation arepresented. Data is expressed as geometric means. P-values for comparisons of these ratios at day14 under budesonide/formoterol (320/9 µg 4 times daily) versus prednisolone (30 mg once daily)and placebo

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Figure 4: Treatment effect on Clinical COPD Questionnaire*On day 3, 7, and 14, the difference of CCQ-score of the visit under study from the CCQ-score atrandomisation are presented. The difference in means from start to end of treatment are significantfor budesonide/formoterol versus prednisolone (p=0.02) Data is expressed as means. P-values forcomparisons of the arithmetic mean changes at day 14 under budesonide/formoterol (320/9 µg 4times daily) versus prednisolone (30 mg once daily) and placebo

Adverse events and safety

No serious adverse events occurred during study treatment. One seriousadverse event (operation for a sinus maxillaris cyst) was reported during thethree months follow-up period. Eighteen patients had a treatment failure (definedas need for open label COPD treatment in the first 3 weeks after start of studytreatment) or relapse (defined as need for open label COPD treatment from 3weeks to 3 months after start of treatment) in the 3 months after start oftreatment (7 in B/F, 7 in PRED, 4 in the PLAC treatment arm). Nohospitalisations for respiratory symptoms were required. Furthermore there wereno different patterns between the treatment groups in adverse events,characteristics on electrocardiograms, or blood pressure and heart rate, thoughunder PRED QRS duration decreased compared to PLAC(-2.7 ms versus +3.6ms, p=0.0048). B/F treatment did not significantly suppress serum cortisolcompared to placebo (mean 16% lower p=0.50). By contrast, the PREDtreatment did suppress serum cortisol levels significantly versus placebo (mean45% lower; p=0.03). There were no significant differences in the changes in theserum glucose levels between the groups. The decrease in serum potassium

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under PRED (-0.29 mmol/L) differed significantly form the increase under PLAC(+0.03 mmol/L, p=0.03).

Table 4. Lung function parameters_______________________________________________________________________________


FEV1 (L), day 1 1.56 (36) 1.57 (28) 1.45 (26)FEV1, (L), day 14 1.68 (35) 1.61 (30) 1.50 (23)IVC, (L) day 1 3.93 (27) 4.27 (25) 3.83 (20)IVC, (L) day 14 3.91 (25) 4.31 (20) 3.74 (21)

FEV1 /VC (%) day 1 40 (27) 37 (26) 38 (24)FEV1 /VC (%) day 14 43 (27) 37 (29) 41 (16)

sGaw, (kPa/L/s) day 1 0.59 (43) 0.62 (49) 0.62 (44)

sGaw, (kPa/L/s) day 14 0.70 (50) 0.67 (42) 0.76 (49)

_______________________________________________________________________________Lung function parameters. Data is expressed as geometric mean and coefficient of variation(sd/mean x 100%). All differences p>0.05. pred= predicted. p-values for comparisons of the ratio atday 14 to day 1 under budesonide/formoterol (320/9 µg 4 times daily) versus prednisolone (30 mgonce daily) and placebo.

Discussion

Treatment of COPD exacerbations with high dose budesonide/formoterol (B/F)significantly reduced sputum eosinophils compared to placebo. This reduction inairway inflammation was accompanied by an improvement in symptoms.Prednisolone (PRED) treatment also reduced airway inflammation andsymptoms. In contrast to B/F, PRED significantly suppressed plasma cortisollevels.

Several studies have reported that eosinophils increase during COPDexacerbations, both in airway biopsies and in induced sputum 15;16;29. Althoughthe effects of steroids (inhaled or oral) on inflammation during exacerbationshad not been previously studied, several studies have assessed the effects ofcorticosteroids on inflammation in stable COPD. Gan et al performed ameta-analysis of the effects of inhaled steroids in the stable phase of COPD,showing a trend towards reducing eosinophil counts in the sputum of stableCOPD patients 30. In a recent study, treatment of stable COPD with inhaledsalmeterol/ fluticasone propionate also reduced sputum eosinophil counts 31.

To evaluate the potential effects of corticosteroids on airway inflammation, wemeasured the sputum cell mRNA expression of several cytokines that play arole in the attraction/survival of eosinophils, our primary endpoint, or may

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contribute to the induction and resolution of COPD exacerbations. We found alarger decrease in IL-5 expression with B/F compared to placebo. Since IL-5 isinvolved in eosinophil growth and differentiation, this reduction fits well with thereduction in sputum eosinophils 32. Under placebo treatment a larger increasefrom day 1 to day 14 was observed in expression of TGF- ß compared to PREDand similar trends, though not significant, compared to B/F. This increase underplacebo may signify an increased inflammation and tissue damage due to thelack of anti-inflammatory treatment. We do not have an explanation for thesmaller increase in HO-1 expression under PRED compared to B/F. We wouldhave expected a decreased expression by both the active treatments, sinceHO-1 is a stress inducible enzyme, and we would have expected a decrease instress stimuli by treatment due to a reduction in inflammation. Perhaps thiseffect has been confounded by other stress factors such as smoking.

The beneficial effects of steroid treatment on parameters of inflammation wereaccompanied by improvements in clinical symptoms. The total symptom scoresas recorded in the daily diaries were better under both B/F and the PREDtreatment, with a significant improvement in cough for B/F and trends inimprovement in sputum production under both B/F and PRED treatment.

This study is the first to use an airway inflammatory parameter as the majorendpoint for treatment efficacy in exacerbations of COPD. A few randomisedcontrolled trials used clinical endpoints to asses the effects of oral prednisoloneon COPD exacerbations 1;2;33;34, and a meta-analysis concluded that it reducestreatment failure and the need for additional treatment, and increases the rate ofimprovement in lung function and dyspnea over the first 72 hours 35.

Maltais et al showed beneficial effects of inhaled budesonide 3, comparing 2mgnebulised budesonide every 6 hours and 30mg oral prednisolone every 12hours, and both treatments improved airflow limitation when compared withplacebo. Although we did not show a statistically significant effect on airflowlimitation between the three groups in our study, a trend was found in thecomparison between B/F and PLAC (see figure 2, p= 0.07). The lack ofsignificance is likely due to the smaller patient numbers in our study (45 versus199) since the magnitude of improvement in FEV1 was similar (125 ml versus100 ml). Our study sample size was based on power calculations with changesin eosinophil percentage as primary end point. Another difference between thestudies is the time point of evaluation, i.e. in our study at 2 weeks afterrandomisation, versus 72 hours in Maltais' study.

A great advantage of B/F therapy above PRED should be the avoidance, or atleast a reduction, of systemic side effects. Several studies have shown thatinhaled corticosteroids have fewer side effects compared to oral corticosteroids.Morice et al investigated the side effects of nebulised budesonide (2 mg twicedaily) with oral prednisolone (30 mg at once daily) in the treatment ofCOPD-exacerbations. They found that prednisolone treatment resulted in lowerurinary corticosteroid metabolites, and lower serum osteocalcin than inhaledtreatment 36. In our study we investigated serum cortisol levels. We found that

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there was no significant suppression of serum cortisol by B/F treatmentcompared to placebo. PRED treatment did suppress serum cortisol levels asexpected and this aspect favours the treatment with B/F over prednisolone.

Our study was not powered to assess equality of clinical effects of the two activetreatment arms. Such a non-inferiority trial would require more patients. Any lackof difference in statistical terms between B/F and PRED in the present studycould therefore be due to a type 2 error and should not be interpreted to signifyequal or similar effects. Nevertheless, since all parameters favoured B/F overPRED, there was no indication of inferiority of B/F versus prednisolone in thissetting.

The use of B/F for the treatment of mild to moderate exacerbations of COPD athome improved both inflammation and symptoms and was well tolerated.Whether B/F treatment is as effective as PRED and is to be favoured B/F overPRED because of less side effects, will require larger-scale efficacy studies. Aninteresting option is the possibility to treat exacerbations at home in an earlierstage of an evolving exacerbation by B/F, as was recently demonstrated to beeffective in the treatment of asthma 4. Whether this earlier institution of effectivetherapy could also prevent in COPD the development of full-blownexacerbations and thereby hospitalisations is an interesting option that should bestudied in future studies.

We conclude that sputum eosinophils and symptoms of patients with mild tomoderate COPD exacerbations can be improved by a 2-week treatment with160 µg budesonide/ 4.5 µg formoterol, 2 doses q.i.d., compared to placebo. Anext study should aim to establish whether treatment of exacerbations at homewith budesonide/formoterol can replace traditional treatment with oralcorticosteroids

Acknowledgements

The authors thank Ibolya Sloots, Brigitte Dijkhuizen, Koos van de Belt, andJanneke Heimweg for all sputum measurements, Marco van der Toorn for themRNA measurements, the lung function department for the many lung functionmeasurements, and Alec Ross for the cortisol measurements (UMC St.Radboud, Nijmegen).

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(12) Llewellyn-Jones CG, Harris TA, Stockley RA. Effect of fluticasone propionate on sputumof patients with chronic bronchitis and emphysema. Am J Respir Crit Care Med 1996;153(2):616-621.

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(13) Wilson SJ, Wallin A, Della-Cioppa G et al. Effects of budesonide and formoterol onNF-kappaB, adhesion molecules, and cytokines in asthma. Am J Respir Crit Care Med2001; 164(6):1047-1052.

(14) Wallin A, Sandstrom T, Soderberg M et al. The effects of regular inhaled formoterol,budesonide, and placebo on mucosal inflammation and clinical indices in mild asthma.Am J Respir Crit Care Med 1999; 159(1):79-86.

(15) Fujimoto K, Yasuo M, Urushibata K et al. Airway inflammation during stable and acutelyexacerbated chronic obstructive pulmonary disease. Eur Respir J 2005; 25(4):640-646.

(16) Mercer PF, Shute JK, Bhowmik A et al. MMP-9, TIMP-1 and inflammatory cells in sputumfrom COPD patients during exacerbation. Respir Res 2005; 6:151.

(17) Pizzichini E, Pizzichini MM, Gibson P et al. Sputum eosinophilia predicts benefit fromprednisone in smokers with chronic obstructive bronchitis. Am J Respir Crit Care Med1998; 158(5 Pt 1):1511-1517.

(18) Brightling CE, Monteiro W, Ward R et al. Sputum eosinophilia and short-term response toprednisolone in chronic obstructive pulmonary disease: a randomised controlled trial.Lancet 2000; 356(9240):1480-1485.

(19) Brightling CE, McKenna S, Hargadon B et al. Sputum eosinophilia and the short termresponse to inhaled mometasone in chronic obstructive pulmonary disease. Thorax 2005;60(3):193-198.

(20) Bathoorn D, Liesker JJW, Postma DS, Bondesson E, Koëter GH, van Oosterhout AJM,Kerstjens HAM. Anti-inflammatory effect of combined budesonide/formoterol treatment inCOPD exacerbations. Proceedings of the American Thoracic Society 2006; 3:A605


(22) Quanjer PH, Tammeling GJ, Cotes JE et al. Lung volumes and forced ventilatory flows.Report Working Party Standardization of Lung Function Tests, European Community forSteel and Coal. Official Statement of the European Respiratory Society. Eur Respir JSuppl 1993; 16:5-40.


(24) Leidy NK, Schmier JK, Jones MK et al. Evaluating symptoms in chronic obstructivepulmonary disease: validation of the Breathlessness, Cough and Sputum Scale. RespirMed 2003; 97 Suppl A:S59-S70.

(25) van der Molen T, Willemse BW, Schokker S et al. Development, validity andresponsiveness of the Clinical COPD Questionnaire. Health Qual Life Outcomes 2003;1(1):13.

(26) Rutgers SR, Timens W, Kaufmann HF et al. Comparison of induced sputum withbronchial wash, bronchoalveolar lavage and bronchial biopsies in COPD. Eur Respir J2000; 15(1):109-115.

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(27) Pizzichini MM, Pizzichini E, Clelland L et al. Sputum in severe exacerbations of asthma:kinetics of inflammatory indices after prednisone treatment. Am J Respir Crit Care Med1997; 155(5):1501-1508.

(28) Kocks JW, Tuinenga MG, Uil SM et al. Health status measurement in COPD: the minimalclinically important difference of the clinical COPD questionnaire. Respir Res 2006; 7:62.

(29) Saetta M, Di Stefano A, Maestrelli P et al. Airway eosinophilia in chronic bronchitis duringexacerbations. Am J Respir Crit Care Med 1994; 150(6 Pt 1):1646-1652.

(30) Gan WQ, Man SF, Sin DD. Effects of inhaled corticosteroids on sputum cell counts instable chronic obstructive pulmonary disease: a systematic review and a meta-analysis.BMC Pulm Med 2005; 5(1):3.

(31) Barnes NC, Qiu YS, Pavord ID et al. Antiinflammatory effects of salmeterol/fluticasonepropionate in chronic obstructive lung disease. Am J Respir Crit Care Med 2006;173(7):736-743.

(32) Takatsu K. Interleukin 5 (IL-5) and its receptor. Microbiol Immunol 1991; 35(8):593-606.

(33) Emerman CL, Connors AF, Lukens TW et al. A randomized controlled trial ofmethylprednisolone in the emergency treatment of acute exacerbations of COPD. Chest1989; 95(3):563-567.

(34) Albert RK, Martin TR, Lewis SW. Controlled clinical trial of methylprednisolone in patientswith chronic bronchitis and acute respiratory insufficiency. Ann Intern Med 1980;92(6):753-758.

(35) Wood-Baker RR, Gibson PG, Hannay M et al. Systemic corticosteroids for acuteexacerbations of chronic obstructive pulmonary disease. Cochrane Database Syst Rev2005;(1):CD001288.

(36) Morice AH, Morris D, Lawson-Matthew P. A comparison of nebulized budesonide withoral prednisolone in the treatment of exacerbations of obstructive pulmonary disease.Clin Pharmacol Ther 1996; 60(6):675-678.

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Chapter 7

Anti-inflammatory effects of inhaled carbonmonoxide in patients with COPD: a pilot study

Erik Bathoorn1, Dirk-Jan Slebos1, Dirkje S Postma1, Gerard HKoeter1, Antoon J.M. van Oosterhout2, Marco van der Toorn2, H.Marike Boezen3, Huib A.M. Kerstjens1

1 Groningen Research Institute for Asthma and COPD (GRIAC),Department of Pulmonology, 2 Laboratory of Allergology andPulmonary Diseases, 3 Department of Epidemiology, UniversityMedical Center Groningen, University of Groningen, theNetherlands.

Accepted by European Respiratory Journal

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ABSTRACT

Background: In vitro and in vivo studies have shown that carbon monoxide(CO) has both anti-inflammatory and anti-oxidant capacities. Since COPD ischaracterised by inflammation and oxidative stress, low dose CO could be oftherapeutic use. Aim: To investigate the feasibility and anti-inflammatory effectsof 100-125 parts per million (ppm) CO inhalation in patients with stable COPD.Methods: Twenty ex-smoking COPD patients, with post- bronchodilatorFEV1>1.20 liter and FEV1/FVC<70% were enrolled in a randomised, placebocontrolled, cross-over study. Effects on inflammation were measured in inducedsputum and blood. Results: CO inhalation was feasible and patients’ vital signswere unaffected. Two hours a day inhalation of low dose CO on 4 consecutivedays led to a maximal individual carboxyhemoglobin of 4.5%. Two exacerbationsoccurred in the CO period. CO inhalation led to trends in reduced sputumeosinophils (median reduction 0.25% point; p=0.07) and improvedresponsiveness to methacholine (median PC20 0.85 versus 0.63 mg/mL;p=0.098). Conclusion: Inhalation of 100-125 ppm CO by patients with COPD ina stable phase is feasible and led to trends in reduction of sputum eosinophilsand improvement of responsiveness to methacholine. Further studies need toconfirm the safety and efficacy in inflammatory lung diseases.

Introduction

Chronic obstructive pulmonary disease (COPD) is characterised by anabnormal inflammatory response to noxious gasses or particles, the mostimportant of which in the Western World is tobacco smoke (1). However,smoking can not explain the whole of COPD, since many non-smokers,especially in third world countries, develop COPD without smoking. Moreimportantly, the inflammatory response continues when smoking has beendiscontinued for a prolonged period of time. Our long-term goal in COPDresearch is to elucidate the origins of the self-perpetuating inflammatoryresponse in susceptible smokers. This knowledge is a prerequisite for noveltherapeutic interventions in a disease with very few effective treatmentmodalities. In this study we hypothesize that endogenous heme oxygenase-1(HO-1) and its downstream product carbon monoxide (CO) are not induced tothe level necessary to protect the lung from COPD development andprogression.

CO is generated endogenously by heme degredation. This degradation iscatalyzed by the enzyme heme oxygenase (HO). Of the two knownenzymatically active isoforms of HO (HO-1 and HO-2), only HO-1 responds toxenobiotic induction. Constitutively expressed in many tissues, HO-2 occurs athigh levels in nervous and vascular tissues and may respond to regulation byglucocorticoids (2). HO-1 is upregulated in case of tissue injury, for exampleduring periods of tissue hypoxia and/or inflammation (3). Both in vitro and invivo studies suggest therapeutic options for the inhalation of CO since it has

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Anti-inflammatory effects of inhaled carbon monoxide in patients with COPD: a pilot study

potent anti-inflammatory and anti-oxidant capacities (4). In vitro, COdownregulates pro-inflammatory cytokines produced by macrophages (5). Invivo studies in several animal species show that CO has a protective functionagainst ischaemic injury, hyperoxic injury, graft versus host reactions, andpulmonary inflammation (6-8).

It has been postulated that a deficiency in the “HO-1 - CO pathway” leads to adecreased lung protection and thus may contribute to the severity of COPD.We subscribe to this hypothesis since the HO-1 expression in alveolarmacrophages in smoking COPD patients is decreased compared to smokerswithout COPD (9). Additionally, we have previously shown a lower HO-1expression in ex-smokers with COPD compared to healthy ex-smokers (10).Also a polymorphism that is linked with the development of COPD may occur inthe promoter region of the HO-1 gene, resulting in a reduced inducibility ofHO-1 (11). Furthermore, it has recently been shown in vivo that adenoviralHO-1 overexpression suppresses emphysema development (12). Thus, agenetically dependent downregulation of HO-1 expression may arise insub-populations, possibly linked to increased susceptibility to oxidative stress(13).

The ongoing inflammation after smoking cessation in COPD patients providesan inflammatory model for investigation of the anti-inflammatory effects oflow-dose inhaled CO, bypassing the disturbing effect of variable carbonmonoxide levels inhaled during cigarette smoking. Whether exogenousadministration of CO reduces the inflammation and oxidative stress caused bythe postulated impairment to generate sufficient CO endogenously in COPDpatients is the key question of this study. If so, inhalation of CO by COPDpatients could become a realistic therapeutic option. In COPD there is muchexperience with the inhalation of medical gas in the form of oxygen both asmaintenance at home and during exacerbations. It would be feasible to add alow concentration of carbon monoxide to this oxygen.

The purpose of the present pilot study was to explore the feasibility and safetyof inhalation of CO by stable COPD patients, as well as its anti-inflammatorypotential. We hypothesise that inhalation of CO reduces the ongoinginflammation in patients with COPD who stopped smoking.

Methods

Pilot

A pilot study was first performed to assess feasibility and safety of the inhalationof CO. CO was administered from a cylinder in a fixed dosage of 100 parts permillion (ppm) in room air (Nederlandse Technische Gasmaatschappij, Tilburg,the Netherlands). A healthy subject inhaled this gas mixture through anon-rebreathing mask with a flow of 10 L/min for 75 minutes. Venouspercentage of carboxyhemoglobin (COHb) was measured every 15 minutes.No adverse effects occurred, and the maximal COHb level was 2.7%.

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Thereafter, 3 patients with stable COPD inhaled 95 ppm carbon monoxide for 2hours a day on 4 consecutive days. During the inhalation sessions patientswere continuously observed and vital signs were monitored. Again, no adverseevents occurred and the maximal venous COHb-level reached was 3.9%. Weconcluded that inhalation of these low concentrations of CO was safe and madeit possible to start with a randomised controlled clinical trial.

Study design

All patients were required to meet the following criteria: diagnosis of COPD > 1year; age 40–85 yrs; completely stopped smoking> 1 year with 10 pack-years;postbronchodilator FEV1 /Forced Vital Capacity (FVC) <70%; no history ofasthma; no other interfering medication or diseases, no upper/lower respiratorytract infection the last 4 weeks. Allowed medications were short-actinganticholinergics and ß2-agonists as needed; allowed in a fixed dose regimenwere theophyllines, inhaled, nasal, or systemic corticosteroids, and othernon-pulmonary medication.The study was approved by the medical ethics committee of University MedicalCenter Groningen (The Netherlands). All participants gave their writteninformed consent.

At the inclusion visit, history was taken and physical examination performed.Spirometry was performed before and after 400 µg salbutamol. Allergic statuswas taken from medical records and history taking. The study was randomised,placebo controlled, and cross-over. Patients inhaled 100-125 parts per millionCO for 2 hours on 4 consecutive days. After minimally one week wash-out roomair was inhaled in the same schedule. The sequence of CO or placebo wasrandomised (figure 1, study design). The inhalation was blinded for the patient,physician, and laboratory technician; lung function technicians were not blindedsince the CO measurements uncovered the blinding totally. Before and after thefirst 2-hour session, heart rate and blood pressure were assessed. Sputum wasinduced according to European Respiratory Society’s guidelines, but withmodifications when the FEV1 was below 1.5 litres (14;15). Whole sputumsamples were processed for cell counts within 120 minutes as described before(16). A total cell count was performed on sputum samples after addition of a0.1% dithiothreitol equal to the sample’s volume and filtration. Viability waschecked by means of trypan blue exclusion. Two slides for differential cellcounts were stained with May-Grunwald-Giemsa. Differential cell counts wereperformed by counting 300 non-squamous cells in a blinded fashion by twotechnicians, and the mean was used for analysis. Percentages were calculated.Airway hyperresponsiveness was measured as the provocative concentration ofmethacholine causing a 20% fall in the FEV1 (PC20) using the 2-minute tidalbreathing method. Health status was measured by the Clinical COPDQuestionnaire (17). After each inhalation session, patients were asked tomention any adverse symptom experienced.

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Blood differential counts were analysed by flow cytometry (Coulter-STKS,Beckman Coulter, Miami, USA) in the routine hospital laboratory. SerumC-reactive protein (CRP) and albumin were measured by nephelometry (DadeBehring, Leusden, the Netherlands).Serum lipid peroxides were measured as malonaldehyde-thiobarbituric acidadducts by fluorimetry (Sigma-Aldrich, Zwijndrecht, the Netherlands).

Per protocol, a statistician independent of the study performed an interimanalysis after the first 10 patients, to determine, based on indications oftherapeutic effects, whether to continue with inhalation of 100 ppm CO orincrease to 125 ppm CO. The trial was continued using 125 ppm CO.

Figure 1: Study design.CO: carbon monoxide inhalation for 2 hours (4 successive days); Pla: placebo inhalation for 2 hours(4 successive days); LF: lung function; PC20: provocative concentration of methacholine causing a20% fall in FEV1. Lung function, sputum and blood were assessed 17 hours after the last inhalationof CO or placebo.

Statistics

Data are expressed as medians (interquartile range). Non-normally distributedparameters were log-transformed if this normalised the distribution. Normallydistributed efficacy parameters were compared by paired T-test, non-normallydistributed parameters by Wilcoxon signed rank test. The primary parameterwas the difference in sputum neutrophil after CO and after placebo inhalation.The power calculation was performed on the primary parameter: the differencein sputum neutrophil counts after CO and after placebo inhalation. Limited datawas available on the course of the disease as assessed by inflammatoryparameters. In the present study the patients served as their own controls duringthe study, and the changes within patients were compared. A pilot study with 32

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COPD patients in the University Hospital Groningen, tested with one-weekinterval showed a mean change in % neutrophil count of 3.0 % points and astandard deviation of the difference of 11.8% points. With 20 patients in thistwo-treatment crossover study, we had an 80% probability to detect a treatmentdifference of at least 8 % with an alpha of 0.05. Effects of treatment order anddose of CO were analysed post-hoc using an ANOVA-model, with the change ineosinophils from placebo to CO treatment as the dependent variable, andinhaled corticosteroids, phenotype and allergy in history as covariates.

Results

Patients

Patient characteristics are shown in Table 1.Eighteen of the 20 enrolled patients completed the study. One patient withdrewhis consent after randomisation but before the start of the first inhalation sessionand was not included in analyses. One patient discontinued the study due to anSAE after completing the CO period (see safety/adverse events below). Onepatient had only a 1-day inhalation program in the second period (placeboperiod), because the time schedule was too intensive for him.

Table 1 Patient characteristics_______________________________________________________________________________ n=19

Sex, male/female 18/1 Age, years* 67 (63-70)Smoked, packyears* 37 (21-69)Duration of smokingcessation (years) 5.7 (4.6-14.6)FEV1, % pred* 69 (55-79)FEV1, litres 2.2 (1.8-2.5)FEV1/FVC* 52 (44-61)Reversibility, % of pred* 10.1 (5.1-12.6)Allergy yes/no 6/13Usage of ICS yes/no 13/6_______________________________________________________________________________*median (interquartile range)ICS: inhaled corticosteroidsFEV1: forced expiratory flow in one secondFVC: forced vital capacityPred: predicted

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Safety and adverse events

The median COHb reached after the 4th inhalation session of 100 ppm CO was2.6%, with a highest individual value of 3.5%. After 125 ppm inhalation themedian COHb was 3.1%, with the highest individual value reaching 4.5% (table2).The adverse events are shown in table 3. During/after the CO inhalation period,2 exacerbations of COPD occurred. The first patient reported increased dyspneaand cough symptoms one week after inhalation of CO, but this did not require achange in medication other than increasing the use of bronchodilators. Twomonths later, a full blown exacerbation occurred, requiring hospitalisation andintubation, which recovered slowly but fully. The second patient had anexacerbation on the third day of CO inhalation, and was treated successfully athome with oral corticosteroids and bronchodilators. There were no differences inother adverse events between CO and placebo inhalation. There were nosignificant differences in change of both heart rate and blood pressure betweenCO and placebo inhalation (table 2).

Table 2 safety parameters

CO Placebo p-valueMedian COHb%* 2.7 (2.1-3.4) 0.2 (0.1-0.7) <0.01Highest COHb%* 4.5 1.7 <0.01Change heart rate (bpm) 0 (-11.3 – 4) -4 (-15 – 0.0) 0.08Change systolic bloodpressure(mmHg)

10 (-1.3 – 20) 10 (0.0 – 20) 0.80

Change diastolic bloodpressure (mmHg)

6.0 (-5.0 – 15) 12.5 (1.3 –20)

0.23

_______________________________________________________________________________

COHb: percentage of hemoglobin attached with carbon monoxide. Median and highest COHb% aremeasured after the fourth inhalation of each round. Changes in vital signs are expressed as thevalue after minus the value before the first inhalation session.

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Table 3 Adverse events_______________________________________________________________________________

CO inhalation: Placebo inhalation:

SAE: (number of events) SAE: (number of events)Exacerbation requiring intensive nonecare, 2 months after CO inhalation: 1

Adverse events: (number of events) Adverse events: (number of events)Exacerbation COPD, on third dayof CO inhalation: 1Headache: 3 Headache: 2Dry mouth: 2 Dry mouth: 1Dizziness: 1 Dizziness: 1Haemoptysis: 1 Painful chest: 1Cough: 1______________________________________________________________________________

SAE: serious adverse event

Effects on inflammatory indices

There was no reduction by CO inhalation in our primary endpoint sputumneutrophil percentage (table 4). However, the sputum eosinophil percentage didshow a trend towards reduction by CO inhalation (p=0.07, table 4; fig 2).In blood, no significant changes in leukocyte numbers (CO: 6.8 compared toplacebo: 6.9, p=0.36), malonaldehyde levels (CO: 13.4 microM compared toplacebo: 14.2 microM, p=0.53), C - reactive protein levels (CO: 2.5 mg/Lcompared to placebo: 2.5 mg/L, p=0.85), or erythrocyte sedimentation rates(CO: 6.0 mm/hour compared to placebo: 7.0 mm/hour, p=0.21) were found.The first 9 patients were treated with 100 ppm CO, the last 10 with 125 ppm.Post-hoc analysis showed that there were no differences in effects between 100and 125 ppm CO on sputum neutrophils, and eosinophils. The randomizedtreatment order was also of no influence on the effects of treatment on theseparameters.

Effects on lung function and health status

CO inhalation resulted in a trend in improvement of responsiveness tomethacholine (median PC20 after CO inhalation 0.85 versus 0.63 after placeboinhalation; p= 0.098) (fig 3). There was no effect of the CO inhalation on FEV1,FEV1/FVC, sGAW, or on the health status as measured by the CCQ (table 5).

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Table 4 Sputum inflammatory indices

CO Placebo Difference* pTotal cells x106/ml 3.3 (1.6-6.1) 5.1 (2.9-10.4) -0.20 (-3.54-1.38) 0.55Neutrophils % 75.7 (64.1-80.4) 73.4 (60.7-80.6) 2.30 (-4.63-8.08 0.34Eosinophils % 0.75 (0.30-1.55) 1.0 (0.65-2.58) -0.25 (-1.05--0.13 0.07Macrophages % 17.8 (13.4-30.2) 19.9 (13.0-33.7) -2.25 (-6.58-2.95) 0.22Lymphocytes % 1.6 (1.1-2.4) 1.6 (1.0-2.8) -0.30 (-1.00-0.55 0.63

Sputum/Blood albuminratio

1.2 (0.8-2.1) 1.5 (1.0-4.6) -0.26 (-0.95-0.33) 0.21

______________________________________________________________________________

Data expressed as medians (IQR). *Difference is the CO effect calculated as CO value minus thecorresponding placebo value.

Figure 2: Change in sputum eosinophilsData presented as individual changes and group medians.

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Figure 3: Change in PC20 methacholineData presented as individual changesand group median. PC20: provocative concentration ofmethacholine causing a 20% fall in FEV1.

Table 5 Clinical parameters

CO Placebo p-valuePC20 Methacholine(mg/ml)

0.85 (0.29 – 3.92) 0.63 (0.28 – 1.71) 0.098

FEV1 (L) 1.8 (1.5-2.3) 1.8 (1.5-2.4) 0.94FVC (L) 3.8 (3.5-4.4) 4.0 (3.4-4.4) 0.44FEV1/FVC (%) 49 (38 – 54) 48 (41 – 53) 0.39sGAW 1/kPa*s 0.55 (0.33 – 1.07) 0.49 (0.35 – 0.75) 0.23CCQ 1.0 (0.6 – 1.6) 0.9 (0.6 – 1.7) 0.88

_______________________________________________________________________________

Data presented as median (IQR). Lung function was measured pre bronchodilator. PC20:

provocative concentration of methacholine causing a 20% fall in FEV1. CCQ: Clinical COPDQuestionnaire (higher numbers signify a worse health status; a difference of 0.4 is the minimalclinical important difference) (30).

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Discussion

This is the first study to explore the therapeutic potential of inhalation of lowdose CO by patients with stable COPD. The inhalation was feasible, and itresulted in trends towards therapeutic effects in reducing sputum eosinophilsand improving the responsiveness to methacholine. The trends found in this pilotthis study are useful for the design and power calculations of further studies ofthis novel pathway in the treatment of COPD.

The first objective of this pilot study was to explore the safety and feasibility ofinhalation of carbon monoxide in patients with stable COPD. Inhalation of CO byhealthy subjects has been applied previously in clinical trials which wereperformed to assess the clearance of COHb after CO intoxication. In the currentstudy, we titrated the dosage of CO at COHb levels below the levels of COHb"achieved" with smoking of 20 cigarettes a day where the 24 hour averageCOHb levels of reach 5.3% on average, with peaks above 6% (18). A protocol of100 ppm CO for two hours has been shown in a previous study in healthy youngmen to lead to COHb levels of approximately 4% (19). Therefore we exploredthe therapeutic effects of CO at this protocol, at doses well within what weexpected to be safe. Indeed, the inhalation of 100 ppm led to a maximal COHblevel of 3.1% in our patients with COPD and the highest COHb level reachedwith 125 ppm was 4.5%, which is in the range of the maximal COHb values wehad expected.To further assess the safety, we measured the vital signs before and after thefirst inhalation session and recorded adverse events. We did not find asignificant effect of the inhaled CO on vital signs. One patient reportedhaemoptysis. This patient had a long history of frequent haemoptysis ofunknown origin before this trial. There were two exacerbations in the COperiods; one patient experienced a COPD exacerbation starting on day 4 of COinhalation, 18 hours after the last inhalation. A severe exacerbation occurred inanother patient, 2 months after the last inhalation. Both patients hadexperienced regular exacerbations in the past, and we speculate but can in noway prove that these problems were not caused by the CO inhalation itself. Thisstudy is underpowered to differentiate with any certainty, whether theseexacerbations occurred coincidentally during/after CO inhalation, or werecaused by inhalation of CO.

The second objective of this pilot study was to explore effects of inhaled carbonmonoxide on airway and systemic inflammation and oxidative stress. Theprimary end-point was sputum neutrophil counts, an endpoint chosen sinceneutrophils are the predominant cells in sputum also after smoking cessationnext to eosinophils (20). Additionally, CO has been shown to reduce neutrophilsin ovalbumin induced airway inflammation in vivo (8;21). The sputum neutrophilsdid not change significantly. Nevertheless, total cell count showed a downwardtrend, but with a large spread. The p-value for the reduction in eosinophilpercentages approached significance (0.07). The reduction in absoluteeosinophil counts yielded the same p-value of 0.07. The reduction in eosinophils

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could have been influenced by the use of inhaled steroids, since steroidssuppress sputum eosinophils in COPD (22). Thirteen patients were on regularinhaled steroids. It is possible that a larger, and significant, reduction in sputumeosinophils would have occurred had these patients not been on maintenanceinhaled steroids. However, post-hoc analyses did not show differences in trendsin effects by CO in inhaled steroid users and non-users. The reduction ineosinophil numbers is not totally unexpected since animal models showed thatinhalation of CO or cigarette smoke containing CO reduces airway and lungeosinophilia (8;21). It is tempting to speculate that next to the many andoverriding deleterious effects of cigarette smoke, certain components might alsoexert beneficial effects. To be clear, to our opinion these potential beneficialeffects of CO (and perhaps of other components of cigarette smoke such asnitric oxide and nicotine) do not sufficiently counterbalance the harmful effects.

We showed a trend (p=0.098) towards improvement in responsiveness tomethacholine by CO. Both the animal models mentioned above showed areduction in pulmonary eosinophils also showed an improvement inresponsiveness to methacholine (8;21). Our study supports this theory thatinhalation of CO reduces eosinophils and improves responsiveness tomethacholine. The improvement in hyperresponsiveness could be caused by areduction of eosinophils, although in an earlier report the correlation betweenresponsiveness to methacholine and sputum eosinophil counts in patients withCOPD did not reach significant levels (?-0.32, p=0.085) (23). It could also becaused by a direct protective effect of CO on the airway smooth muscles, sinceCO is also a neurotransmitter causing bronchodilation in the airways via cyclicGMP (24).

There are two important methodological issues to discuss. The most importantis that the study was not designed to detect significant changes in sputumeosinophils and responsiveness to methacholine. Post-hoc power analysisshowed that we would have needed 31 patients to cause the same medianeffect size of 0.25% point reduction in the present study to be significant with80% power and an alpha of 0.05. The second is that we did not have a baselinemeasurement before the second phase of the cross-over. However, we foundno evidence of a period effect (carry over effect) in our ANOVA analysis. Thewash-out period of at least a week therefore indeed seems long enough.

The lack of overall significant changes could be due to several reasons. We hadlittle reference for the concentration, duration and frequency of CO inhalation tostart with. The COHb half time is approximately 340 minutes (19). Therefore,inhaled CO at the concentrations we used is largely cleared in 24 hours, whichmakes repetitive inhalation of CO on consecutive days non-cumulative andtherefore feasible. For safety reasons, we preferred to start with a lowconcentration carbon monoxide and divided the exposition over a week. Toinvestigate whether these choices of dose and duration and frequency led to theexpected COHb levels and therapeutic results in the trial, a statisticianindependent of the study performed an interim analysis after the first 10 patientshad completed the trial. Based on predefined criteria, she determined tocontinue the study using 125 ppm. Post-hoc analysis did not show any trends of

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larger effects by inhalation of the higher dose; however, the groups are small.The concentration we used was cautious, and there is margin for inhalation ofhigher concentrations. Other schedule options as a shorter exposition withhigher concentrations, a several-times-daily exposition, or more consecutivedays could lead to different results. Intuitively, one would opt for highercumulative doses. However, it is not a given fact that higher concentrationswould have larger effects. The concentrations of CO we used are already about20-fold higher than physiological alveolar levels as measured in exhaled air, andcarbonyl stress might counteract the anti-inflammatory effects (25).Nevertheless, in an in vitro study a positive dose-response effect was observed:larger anti-inflammatory effects by higher CO concentrations in a dose rangefrom 50-500 ppm (26). The optimal scheme and concentration of CO inhalationneeds to be elucidated in future trials.

If the hurdles of optimal dosing and timing of CO inhalation can be overcome, itwould also be interesting to speculate about other indications than stable COPD,such as COPD exacerbations and severe asthma. Several studies have shownthat both inflammation and oxidative stress are more increased during COPDexacerbations than in stable COPD, with concomitant upregulation of HO-1(27-29). Severe asthma would be another tempting indication for CO treatmentto explore for its allergenic component since CO has been proven to beanti-inflammatory in models of allergen-induced inflammation (8). In addition tothe anti-inflammatory capacities, the bronchodilating capacity of CO makespulmonary diseases an attractive field to explore for indications.

We conclude that inhalation of low dose CO by patients with stable COPD is welltolerated, feasible and safe with respect to the peak COHb levels reached andthe lack of effects on hemodynamics. Whether there is some increased risk ofexacerbations remains to be determined. Inhalation of 100-125 ppm CO led totrends towards reduction in sputum eosinophils and improvement of bronchialresponsiveness. This indicates that inhaled CO might have therapeutic effects inCOPD. With these data, future studies should define more optimal schemes anddoses, and assess the anti-inflammatory and anti-oxidative stress as well as thepotential therapeutic capacities of CO inhalation.

Acknowledgements

The authors would like to thank Ibolya Sloots, and Brigitte Dijkhuizen of theLaboratory of Allergology and Pulmonary Diseases, Jacco Zwaagstra of theSurgery Research Laboratory, Dr J.M.Vonk of the Epidemiology Department, DrN.H.T. ten Hacken of the Pulmonary Department, and the Lung FunctionDepartment for all the lung function measurements, all from the UniversityMedical Center Groningen.

Supported by a research grant from Stichting Astma Bestrijding, theNetherlands. An unrestricted grant was received from AstraZeneca theNetherlands for salary support of E. Bathoorn.

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(6) Fujita T, Toda K, Karimova A, Yan SF, Naka Y, Yet SF et al. Paradoxical rescue fromischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis.Nat Med 2001; 7(5):598-604.

(7) Sato K, Balla J, Otterbein L, Smith RN, Brouard S, Lin Y et al. Carbon monoxidegenerated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiactransplants. J Immunol 2001; 166(6):4185-4194.


(9) Maestrelli P, Paska C, Saetta M, Turato G, Nowicki Y, Monti S et al. Decreased haemoxygenase-1 and increased inducible nitric oxide synthase in the lung of severe COPDpatients. Eur Respir J 2003; 21(6):971-976.

(10) Slebos DJ, Kerstjens HA, Rutgers SR, Kauffman HF, Choi AM, Postma DS. Haemoxygenase-1 expression is diminished in alveolar macrophages of patients with COPD.Eur Respir J 2004; 23(4):652-653.

(11) Yamada N, Yamaya M, Okinaga S, Nakayama K, Sekizawa K, Shibahara S et al.Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated withsusceptibility to emphysema. Am J Hum Genet 2000; 66(1):187-195.

(12) Shinohara T, Kaneko T, Nagashima Y, Ueda A, Tagawa A, Ishigatsubo Y.Adenovirus-mediated transfer and overexpression of heme oxygenase 1 cDNA in lungsattenuates elastase-induced pulmonary emphysema in mice. Hum Gene Ther 2005;16(3):318-327.

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(13) van Beurden WJ, Smeenk FW, Harff GA, Dekhuijzen PN. Markers of inflammation andoxidative stress during lower respiratory tract infections in COPD patients. Monaldi ArchChest Dis 2003; 59(4):273-280.

(14) Djukanovic R, Sterk PJ, Fahy JV, Hargreave FE. Standardised methodology of sputuminduction and processing. Eur Respir J Suppl 2002; 37:1s-2s.

(15) Pizzichini MM, Pizzichini E, Clelland L, Efthimiadis A, Mahony J, Dolovich J et al. Sputumin severe exacerbations of asthma: kinetics of inflammatory indices after prednisonetreatment. Am J Respir Crit Care Med 1997; 155(5):1501-1508.


(17) van der Molen T, Willemse BW, Schokker S, ten Hacken NH, Postma DS, Juniper EF.Development, validity and responsiveness of the Clinical COPD Questionnaire. HealthQual Life Outcomes 2003; 1(1):13.

(18) Zevin S, Saunders S, Gourlay SG, Jacob P, Benowitz NL. Cardiovascular effects ofcarbon monoxide and cigarette smoking. J Am Coll Cardiol 2001; 38(6):1633-1638.

(19) Peterson JE, Stewart RD. Absorption and elimination of carbon monoxide by inactiveyoung men. Arch Environ Health 1970; 21(2):165-171.

(20) Rutgers SR, Postma DS, ten Hacken NH, Kauffman HF, Der Mark TW, Koeter GH et al.Ongoing airway inflammation in patients with COPD who do not currently smoke. Thorax2000; 55(1):12-18.

(21) Melgert BN, Postma DS, Geerlings M, Luinge MA, Klok PA, van der Strate BW et al.Short-term smoke exposure attenuates ovalbumin-induced airway inflammation in allergicmice. Am J Respir Cell Mol Biol 2004; 30(6):880-885.

(22) Perng DW, Wu CC, Su KC, Lee YC, Perng RP, Tao CW. Inhaled fluticasone andsalmeterol suppress eosinophilic airway inflammation in chronic obstructive pulmonarydisease: relations with lung function and bronchodilator reversibility. Lung 2006;184(4):217-222.

(23) Willemse BW, ten Hacken NH, Rutgers B, Lesman-Leegte IG, Timens W, Postma DS.Smoking cessation improves both direct and indirect airway hyperresponsiveness inCOPD. Eur Respir J 2004; 24(3):391-396.

(24) Cardell LO, Ueki IF, Stjarne P, Agusti C, Takeyama K, Linden A et al. Bronchodilatationin vivo by carbon monoxide, a cyclic GMP related messenger. Br J Pharmacol 1998;124(6):1065-1068.

(25) Paredi P, Kharitonov SA, Leak D, Ward S, Cramer D, Barnes PJ. Exhaled ethane, amarker of lipid peroxidation, is elevated in chronic obstructive pulmonary disease. Am JRespir Crit Care Med 2000; 162(2 Pt 1):369-373.

(26) Morse D, Pischke SE, Zhou Z, Davis RJ, Flavell RA, Loop T et al. Suppression ofinflammatory cytokine production by carbon monoxide involves the JNK pathway andAP-1. J Biol Chem 2003; 278(39):36993-36998.

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(28) Rahman I, Skwarska E, MacNee W. Attenuation of oxidant/antioxidant imbalance duringtreatment of exacerbations of chronic obstructive pulmonary disease. Thorax 1997;52(6):565-568.

(29) Tsoumakidou M, Tzanakis N, Chrysofakis G, Siafakas NM. Nitrosative stress, hemeoxygenase-1 expression and airway inflammation during severe exacerbations of COPD.Chest 2005; 127(6):1911-1918.

(30) Kocks JW, Tuinenga MG, Uil SM, van den Berg JW, Stahl E, van der Molen T. Healthstatus measurement in COPD: the minimal clinically important difference of the clinicalCOPD questionnaire. Respir Res 2006; 7:62.

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Summary

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Summary

This thesis describes the results of two clinical trials: the Symbexco trial, inwhich the anti-inflammatory effects of inhaled budesonide/formoterol intreatment of COPD exacerbations were investigated, and the “CO in COPD”trial, in which the anti-inflammatory capacity of inhaled carbon monoxide instable COPD was explored.

In Chapter 2, we reviewed the literature on airways inflammation and treatmentof exacerbations of COPD. We found that the sputum counts of eosinophils,neutrophils, and lymphocytes are increased during COPD exacerbations, andthat there are associations between the cell type that is increased and the causeof the exacerbation. Eosinophils are more increased in exacerbations caused bya virus, whereas neutrophils are more elevated in exacerbations of bacterialaetiology. Lymphocytes seem to play an important regulatory role inexacerbations; however, literature so far has provided little insight in the preciseinflammatory mechanisms.

We reviewed the anti-inflammatory drugs that were studied for their efficacy intreating COPD exacerbations. We concluded that only oral steroids have beenproven successful until now. However, the effects of oral steroids are modest,and oral steroids do have important systemic side effects. The use of inhaledsteroids (in combination with long acting bronchodilators) has been reported asan alternative to systemic steroids in the treatment of exacerbations with lesspotential for systemic side effects. For the future treatment of COPD, we foundthat several more drugs aiming at novel targets are in development for stableCOPD. A further step will be to test some of these drugs also in COPDexacerbations.

We concluded in our review that further research is required to more fullyunderstand the inflammatory mechanisms in the onset and development ofCOPD exacerbations. This may make inflammatory pathway-specificinterventions possible, resulting in a more effective treatment of COPDexacerbations with fewer side effects.

In Chapter 3, we described the results of our evaluation of whether the inductionof sputum is safe during COPD exacerbations. Sputum induction is a validatedmethod to acquire information on airway inflammation. During sputum induction,patients inhale nebulised saline of various concentrations, by which thecoughing-up of sputum is facilitated. Furthermore, the samples gained bysputum induction are of a better quality compared to spontaneously producedsputum.Unfortunately, some patients develop a bronchoconstrictive reaction to inhaledsaline. Therefore the forced respiratory volume in 1 second (FEV1) of eachpatient was monitored after each step of saline inhalation during the sputuminduction. With this close monitoring, sputum induction is generally consideredsafe, even in patients with COPD with a severe airway obstruction. To the bestof our knowledge, the safety of sputum induction during exacerbations of COPD

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had however not been assessed. To do so, we analysed our data on safety andbronchoconstriction during exacerbations, and compared this to the data of thesame patients during sputum induction in a stable phase.

We found that considerable decreases in FEV1 can occur, but they aresustained well. The decreases in FEV1 by sputum induction during anexacerbation are of similar absolute magnitude or even smaller than in thestable phase. Monovariate analysis showed that a larger decrease in FEV1 dueto sputum inductions during exacerbations can be predicted by a largerdecrease in FEV1, a lower sputum total cell count, and a higher eosinopihilpercentage in the induced sputum differential count during the stable phase ofCOPD. The sole independent predictor of the fall in FEV1 during sputuminduction in COPD patients with an exacerbation was the decrease in FEV1during sputum induction during a stable phase. We concluded that induction ofsputum in mild to moderate exacerbations of COPD can be performed as safelyas during stable phase, using a cautious protocol in patients with severe COPD.

In Chapter 4, we studied the effects of steroid withdrawal in patients with COPDin a stable phase. In the Symbexco trial, inhaled steroids were withdrawn formethodological reasons: we wanted to avoid that the anti-inflammatory effects ofinhaled steroids influenced the baseline measurements. Two months after thesteroid withdrawal, we performed baseline measurements. We noticed thatmany patients did not sustain this withdrawal well: their FEV1 decreased, andquality of life worsened. This has been reported in other trials. We investigatedwhich inflammatory mechanisms were associated with the worsening of FEV1and quality of life. We found that sputum eosinophils were increased aftersteroid withdrawal, and that this increase was associated with the worsening ofFEV1 and quality of life.

In Chapter 5, we studied the change in inflammation from a stable phase ofCOPD to an exacerbation. We found a general increase in airway inflammationduring COPD exacerbations: sputum eosinophil, lymphocyte, and neutrophilnumbers all rose compared to the stable phase of the disease.

There are various causes which lead to exacerbations of COPD: viral, orbacterial airway infections, or exposure to air pollution have been identified ascauses. We investigated whether these causes are associated with differentinflammatory cellular patterns. We found that some inflammatory markers,specifically sputum LTB4, MPO, IL-6, TNF-a, and serum CRP and IL-6, whichare commonly associated with exacerbations, are increased during bacterialexacerbations, but little or not increased at all during non-bacterialexacerbations.

It is important to identify a bacterial cause of exacerbation, since theseexacerbations should be treated with antibiotics. Sputum cultures are often usedin the decision to prescribe antibiotics. The sputum gram staining, which can bedetermined immediately, is often not specific enough for this purpose, whereas

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waiting for the results of sputum culture takes about 5 days, which is too long.Therefore inflammatory markers, if specific and sensitive for a bacterial airwayinfection, would be of clinical use. Using receiver operated curves, wedetermined the predictive values of those markers we that we had found to beincreased in bacterial infections. We concluded that sputum TNF-a has the bestpredictive values to differentiate a bacterial infection from a non-bacterial causeof exacerbation.

In Chapter 6, we described the results of a study of the treatment ofexacerbations: the Symbexco study. COPD exacerbations are generally treatedwith oral steroids and increased use of bronchodilators. As described earlier inthe summary of Chapter 2, the effects of oral steroids in COPD exacerbationsare modest, and come with considerable systemic adverse effects. It would be astep forward if we could treat patients with steroids avoiding these systemicadverse effects. Inhaled steroids have been reported to cause less systemiceffects. Therefore, we explored the anti-inflammatory and therapeutic effects ofinhaled budesonide/formoterol. We compared the effects of inhaledbudesonide/formoterol to placebo, and to oral prednisolone, primarily on sputumeosinophilic inflammation during COPD exacerbations. Furthermore, wecompared effects of these treatments on lung function parameters, respiratorysymptoms, quality of life, treatment failure and adverse events.

We found that treatment of COPD exacerbations with high dosebudesonide/formoterol reduced sputum eosinophils compared to placebo andresulted in an improvement in respiratory symptoms. Prednisolone treatmentalso reduced airway inflammation and respiratory symptoms. Treatment withprednisolone resulted in a suppression of plasma cortisol levels. Our study wasnot designed to determine whether budesonide/formoterol treatment is aseffective as prednisolone treatment, which would require a larger studypopulation. We concluded that future studies should prove this non-inferiority,before the standard prednisolone treatment can be replaced with inhaledbudesonide/formoterol treatment.

In Chapter 7, we explored the anti-inflammatory effects of inhaled low dosecarbon monoxide. Both in vivo and in vitro studies have shown stronganti-inflammatory effects of carbon monoxide. Next to this, carbon monoxidehas relaxing effects on the airway smooth muscles, since it is a neurotransmittervia cyclic GMP causing bronchodilation in the airways. Additionally, carbonmonoxide is a powerful anti-oxidant. Since inflammation, oxidative stress, andairway obstruction are important features in COPD, we decided to explore thetherapeutic capacity of carbon monoxide in this disease.

The first objective of the study was to assess safety and feasibility of inhalationof low dose carbon monoxide by COPD patients. We titrated the dosage ofcarbon monoxide at carboxyhemoglobine (COHb) levels below that "achieved"by smoking 20 cigarettes a day where the 24 hour average COHb levels reach5.3%, with peaks above 6%. A protocol of 100 ppm CO for two hours had beenshown to lead to COHb levels of approximately 4%. Therefore we explored thetherapeutic effects of CO. The inhalation of 100 ppm led to a maximal COHb

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level of 3.1% in our patients with COPD and the highest COHb level reachedwith 125 ppm was 4.5%, which is in the range of the maximal COHb values wehad expected.

We did not find a significant effect of the inhaled CO on vital signs. One patientreported haemoptesis. This patient had a long history of frequent haemoptesisof unknown origin before this trial. There were two exacerbations in the COperiods; one patient experienced a COPD exacerbation starting on day 4 of COinhalation, 18 hours after the last inhalation. A severe exacerbation occurred inanother patient, 2 months after the last inhalation. Both patients hadexperienced regular exacerbations in the past. We concluded that inhalation oflow dose carbon monoxide is feasible. Future studies should assess whether theadverse events during treatment with carbon monoxide were unrelated to theCO inhalation, which we speculate, or caused by the CO inhalation itself.

The second objective of this pilot study was to explore effects of inhaled carbonmonoxide on inflammation. We found that inhalation of low dose carbonmonoxide results in a trend of reduction of airway inflammation, specificallyeosinophils, and responsiveness to methacholine. We conclude that our data onthese effects are very useful to design larger studies, which may confirm thetrends we found.

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Discussion

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Steroid withdrawal in clinical trials

In chapter 3, we described that withdrawal of inhaled corticosteroids (ICS) inpatients with stable COPD results in an increase in inflammation and airwayobstruction. We furthermore explored the inflammatory mechanisms possiblyresponsible for these effects. These detrimental effects occurred even when thepatients seemed clinically stable since patients experiencing an exacerbationwere treated accordingly and left out of this analysis. These effects of steroidwithdrawal have been described in several studies (1;2), but there is little insighton what causes this deterioration. We found that particularly sputum eosinophilsincreased after steroid withdrawal, which was accompanied by worsening ofairway obstruction and quality of life.

Many studies have a run-in phase, in which ICS are withdrawn to avoid the biasthat the baseline airway inflammation is variably modified by different ICS usage.New trials should reconsider this approach, for several reasons. Firstly,withdrawal by itself also induces bias: withdrawal of ICS induces differences inbaseline inflammation, as not all patients respond the same to ICS withdrawal.Secondly, many patients exacerbate soon after withdrawal. Frequently, thesepatients are then excluded from the trial, resulting in a selection bias of patientstowards a group with less tendency to deteriorate after ICS withdrawal. A thirdconsideration is the dilemma whether it is ethical stop ICS with a subsequentincreased risk of exacerbations for the sake of a study. These patients becomemore symptomatic, and may need additional (systemic) steroid treatment withassociated potential side effects. For real life (not experimentally induced)exacerbations it has been demonstrated that patients sometimes do not fullyrecover form exacerbations to pre-exacerbation levels particularly for peakexpiratory flow values (3).

ICS withdrawal is applied to obtain equal baseline values in COPD patients, andas mentioned above, this is a pitfall. Should we then investigate other ways thatavoid steroid withdrawal? Reduction of steroid treatment in all patients to asimilar low dose maintenance treatment may be a solution to this problem. Butagain a similar dose of ICS may have different effects in an individual patientwith COPD. Another option is a gradual withdrawal of steroids, which mightresults in less deterioration of inflammation and respiratory symptoms. Otherways around this problem would be stratification for baseline steroid use, oreven an increase in steroid use to a high dose for all study participants. Futurestudies should investigate if these options improve the methodology of trials andreduces symptoms for patients.

Non-invasive measurement of inflammation during COPD exacerbations

In chapter 4 we showed that it is safe and feasible to study the cellular patternsof airway inflammation by sputum induction using a hypertonic saline solution

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even during acute exacerbations of COPD. We assessed the safety in mild tomoderate exacerbations that did not require hospitalisation. Sputum inductionshave been assessed in several settings, but there is little data on safety (4-6).These studies did not all use the same induction protocol (i.e. duration,concentration and number of nebulised saline inhalations), which makes itdifficult to compare both the safety and the results of the various studies onairway inflammation during COPD exacerbations. It would be worthwhile to studythe various protocols systematically, in order to reach consensus on a universalprotocol for sputum induction during COPD exacerbations with the bestproperties with respect to safety and a reproducible reflection of the cellularairway inflammation.

Not only inflammatory cells, but also non-cellular biomarkers provide informationon airway inflammation. Thus far, cytokines have given us insight in themechanisms that play a role in COPD in a stable phase and duringexacerbation. However, the measurement of cytokines in sputum of COPDpatients plays little to no role in current clinical practice. We foresee a potential future role for measurement of cytokines during COPDexacerbations in the decision to prescribe antibiotics: in chapter 5, we haveshown that sputum biomarkers might be useful to distinguish a bacterial from anon-bacterial origin of exacerbation. C-reactive protein (CRP) and procalcitoninare the most elaboratively investigated markers for this purpose. CRP has beenevaluated as a marker to define an exacerbation, but was only useful incombination with a major exacerbation symptom. Procalcitonin-guided therapyhas been shown to reduce antibiotics prescription, however the marker does nothave ideal predictive characteristics of bacterial infections in COPDexacerbations, since both its specificity and sensitivity appear to be poor (7-9).From our data, sputum tumor necrosis factor-a seemed to be a potential markerto identify a bacterial cause of exacerbation. However, our study population wasnot large enough to provide firm data on its usefulness or on the best cut-offpoint. Nevertheless, if the favourable predictive value for a bacterial infection ofsputum tumor necrosis factor-a levels could be confirmed in a large study, thismight lead to a novel objective guide for administering antibiotics in COPDexacerbations, perhaps in conjunction with other parameters such asprocalcitonin.

In chapter 5, we studied the relationship between the cause of an exacerbationand the inflammatory pattern. In 7 out of 28 exacerbations we identified abacterial infection as the cause. Viral and bacterial infections and air pollutionare well known causes of exacerbations (10;11). Nevertheless, in up to 30 % ofexacerbations, the cause remains unknown (12). Perhaps these causes remainunknown due to limitations in our ability to detect viruses, or due to our lack ofunderstanding of other causes. Identification of these causes would lead to abetter understanding of the inflammation associated with of COPDexacerbations, and explain why some patients do not respond to conventionalsteroid therapy. It might also open windows to new pathways of treatment.

Gastro oesophageal reflux (GER) might be such an unidentified cause. GER iswell known to cause respiratory symptoms. In asthma, GER is a cause of

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increased respiratory symptoms, which responds well to anti-reflux treatment(13). These increased symptoms are thought to be attributable to aspiration ofgastric acid, causing a neutrophilic inflammation in the airways (14). There islittle research on the role of GER in COPD. Nevertheless, a few studies haveshown that GER is more prevalent in COPD patients than in controls andfurthermore a trend has been observed towards more severe COPD inindividuals in whom GER exists (15-17). An increased prevalence of GER inCOPD patients has been shown by reflux questionnaires and by oesophagealpH measurement (15;18).

At present, it is not known if an episode of increased GER can cause anexacerbation in COPD patients. However, patients with GER do have a muchhigher exacerbation rate compared to patients without GER, suggesting acausative role for GER in COPD exacerbations (19). In vitro research shows thatexposure of bronchial epithelial cells with reflux, and particularly the stomachfluid component pepsin, an enzyme involved in protein break-down, inducesrelease of interleukin -6 and interleukin-8, pro-inflammatory cytokines reportedto be increased during COPD-exacerbations (figure 1). In summary, it seemsimportant to ask patients with COPD about GER symptoms in clinical practice.

Treatment of COPD exacerbations with inhaled budesonide and formoterol.

In chapter 6, we analysed the effectiveness of budesonide/formoterol (B/F) inthe treatment of COPD exacerbations. The treatment with B/F compared to oralprednisolone seemed to have similar effects on airway eosinophils andsymptoms, and even better effects on both airflow limitation and health status.These encouraging results are to be confirmed in future large trials.Furthermore, we studied the outpatient treatment of mild to moderate COPDexacerbations. Whether the effects are the same in hospitalised patients withmore severe exacerbations is an interesting and important research and clinicalquestion to be studied in future trials. To answer, at least in part, these questionsraised from the Symbexco trial, a large multicenter trial has already started(ClinicalTrials.gov Identifier: NCT00259779). We look forward to the results ofthese trials, since they might confirm our results of positive effects with B/Finstead of prednisolone in the treatment of COPD exacerbations.

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Figure 1: Induction of IL-8 release by pepsin: effect of pepsin dose and pH.Interleukin-8 was corrected for cell proliferation and expressed as a % of exposure with thecorresponding pH without pepsin (control). There is a difference in interleukin-8 release induced bypepsin between the pH levels (F=5.1; p<0.01). Interleukin-8 release induced by pepsin is higher atpH 1.5 compared to pH 2.5 (mean difference 221%; p<0.01). Data expressed as mean (histograms)and SEM (bars) for 3 replicates.

It would be an interesting option to treat exacerbations at home at the onset ofan evolving exacerbation by B/F. We hypothesize that treatment with anincreased dose B/F, which the patients already might use as maintenancetherapy, would reduce the delay of steroid treatment which frequently occurswith oral steroids. The early onset of steroid treatment, which is facilitated by thistreatment strategy, might reduce the exacerbation severity, or even prevent anfull-blown exacerbation In asthma, this treatment strategy improves the time tothe first exacerbation, respiratory symptoms, and lung function, and reduced therate of severe exacerbations (20). Figure 2 shows a suggestion for a simpledouble-blind randomised controlled trial design to assess whether the earlyincrease of B/F treatment could prevent the development of full-blownexacerbations leading to hospitalisations. In this trial patients would be includedin a stable phase, and all patients would receive B/F maintenance treatment intheir stable phase. Patients would be randomised to use increased dose B/F or

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placebo for two weeks at the onset of increasing symptoms, with additional shortacting bronchodilators as needed in both groups. The patients should beinstructed to contact the physician when a full blown exacerbation develops,requiring open label prednisolone treatment. The rescue use of prednisolonewould be the (primary) endpoint of the study. If health improves, the patientscontinue their maintenance treatment after the 2 weeks of add-on trial treatment.During each subsequent period of increased symptoms the trial medicationwould again be used, until there is a need for open label prednisolone treatment,or 1000 days have passed, whichever comes first. During baseline and the trialtreatment periods, symptoms and quality of life should be recorded, and at theend of baseline and treatment periods lung function, adverse symptoms relatedto steroid treatment, and responsiveness to methacholine would be measured.These would be analysed as secondary endpoints (need for open labelprednisolone is the primary endpoint, as mentioned above).

Figure 2: Study design to investigate effectiveness of increasing B/F maintenance dose in theprevention of full blown COPD exacerbation*All patients receive maintenance therapy B/F in adose of 200µg /6µg, twice daily, and shorting acting bronchodilators as needed. ‡ All patientscontinue to use the maintenance therapy. The patients randomised for B/F treatment use theinhaler containing the additional B/F in a dose of 200µg/6µg four times daily; the patientsrandomised for placebo use the placebo-inhaler four times daily.

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Inflammatory cells to measure treatment outcome.

In the SYMBEXCO-study, described in chapter 6, we used sputum eosinophilsas the primary endpoint to measure the outcome of treatment effect by steroids.We hypothesized that this inflammatory cell would be a valid endpoint forseveral reasons.

The first reason for choosing eosinophils as primary endpoint, is that variousstudies have found eosinophils to be increased during COPD exacerbations, assummarised in table 2 of the review of chapter 2 (4;5;21;22). We confirmed thisincrease (chapter 5). Although all these studies and we found an increase inairway eosinophil counts during exacerbations, this does not necessarily meanthat eosinophils are actively involved in the increase in respiratory symptomsduring COPD exacerbations. Causality has not been found so far, but our studyprovided some contributing evidence for this: we found a correlation between theincrease in eosinophils form stable phase to exacerbation and a decrease inFEV1, although the relationship was weak. However, we did not find arelationship between improvements in FEV1 and health status and decrease insputum eosinophils during the trial treatment. This does not necessarily rule outa role for eosinophils during COPD exacerbations: The lack of associationbetween change in sputum eosinophils and other treatment effects as airwayobstruction and health status might be explained by the limited number ofpatients in our study, and a third of these patients did not receive steroidtreatment, but placebo. Therefore we might not have had the power to showsuch a relationship. A second explanation might be that eosinophils are notinvolved in all COPD exacerbations, but in a subgroup. Papi et al showed thateosinophils were only increased in exacerbations with a viral infection(4), andour data might be suggestive for an increase in eosinophils in a subgroup aswell (figure 3).

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Figure 3: Increase in sputum eosinophils from a stable phase on varying types and dosages ofinhaled steroids (visit 1), via steroid withdrawal for 2 months, to the beginning of a COPDexacerbation. Sputum eosinophil numbers are significantly increased during the exacerbationcompared to the numbers at the inclusion visit. However, there is a large group of patients withCOPD, in which sputum eosinophils are at a low numbers during an exacerbation, indicating thateosinophils increase only in a subgroup of patients.

The second reason to choose eosinophils as primary endpoint is based onreports showing that sputum eosinophils are responsive cells to steroids.Several studies reported that patients with COPD in a stable phase and higheosinophil levels in sputum had better outcomes in FEV1 and symptoms whentreated with steroids, either orally or inhaled (23-25). In COPD exacerbations,outcomes of steroid treatment depending on sputum eosinophil levels have notbeen tested. An interesting question would be whether patients with loweosinophil numbers in sputum benefit from steroid treatment at all, in stablephase or exacerbation. To study this, the large COPD exacerbation trialsinvestigating the effects of steroid treatment, on which the evidence of steroidtreatment during exacerbations is based, should be repeated,this time withmeasurement of the inflammatory characteristics of patients.

We could also have chosen airway neutrophil counts to assessanti-inflammatory effects of B/F during COPD exacerbations: airway neutrophilcounts are increased during COPD exacerbations, as summarised in table 1 ofthe review (chapter 2). In chapter 5 we confirmed that neutrophil numbers are

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increased during COPD exacerbations. Additionally, neutrophilic inflammationhas been related to airflow limitation and severity of symptoms (4;26). Therefore,inhibition of neutrophil recruitment might be a strategy to treat COPDexacerbations. Leukotrienes are important neutrophil-recruiting cytokines duringCOPD exacerbations (27-29). Therefore inhibition of leukotriene activity couldcause a reduction in neutrophils. Indeed, the treatment of patients with stableCOPD with a leukotriene receptor antagonist resulted in reduction in neutrophils,and improvement in symptoms, quality of life and airway obstruction (30). Sincewe and others found that neutrophils are especially increased in exacerbationscaused by bacterial infections, and neutrophils are known to be involved in theeradication of bacteria (27;31), the investigation of treatment of COPDexacerbation with “anti-neutrophil” therapy should be instituted under strictsupervision and we would recommend anti-bacterial protection during suchtrials, to reduce the chance of bacterial overgrowth and pneumonia.

Future anti-inflammatory therapies in COPD exacerbations: inhalation of carbonmonoxide?

In this thesis, we described two intervention trials with inflammation modifyingdrugs: budesonide/formoterol and inhaled carbon monoxide. We presumed thatreduction of inflammation is beneficial and decreases symptoms. This wasbased on reports of trials in a stable phase, which showed that steroid therapy isbeneficial in patients with a higher sputum eosinophil level (23;25), and that thereduction of eosinophils is the pathway by which steroids reduce airwayobstruction in COPD (24). The effects of budesonide/formoterol were veryencouraging, and when the indications of the treatment effects of our pilot studycan be confirmed in a larger trial, this would shape the future of home treatmentof COPD exacerbations.

The use of inhaled carbon monoxide is not so close to clinical treatment ofCOPD exacerbation yet: we started to explore the effects of carbon monoxide instable COPD population (chapter 7), which –as far as we are aware- was thefirst trial in humans to study the beneficial effects of carbon monoxide in COPD.We did not find significant effects on sputum neutrophil counts, the primaryoutcome parameter; although the median sputum neutrophil count was muchlower (median 2.6 compared to 4.0 x106/ml) after CO treatment compared toplacebo, the variability in our small study population was too large, resulting ininsignificant p-values. We did find a trend of reduction in eosinophils byCO-inhalation. In vivo studies have shown that this anti-inflammatory effect ofcarbon monoxide is caused by inhibition of the mitogen activated protein(MAP)-kinase pathway. MAP-kinase pathways are a group of pathways whichhave in common that they are involved in the signal transduction from anexternal inflammatory stimulus to an inflammatory response of the cell, byactivating intracellular transcription factors of pro-inflammatory cytokines (32).

To explore which anti-inflammatory mechanisms are affected by CO inhalationin humans, we performed additional measurements of the cytokines

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8-isoprostane (figure 4), IL-1ß, IL-5 IL-6, IL-8, IL-10, vascular epithelial growthfactor, and tumor necrosis factor-a. We were unable to confirm the effects oncytokines by CO found in in vitro and in vivo studies: none of these cytokinelevels were significantly reduced by CO inhalation. Possible explanations for thislack of effects on these cytokines could be that the dose of CO was too low, orthat our “inflammatory model”, the ongoing inflammation in COPD patients whowere not current smokers, provided insufficient levels of the inflammatorycytokines to measure effects of CO.

Figure 4. Effects of carbon monoxide on the levels of 8-Isoprostane (urine).

Future studies on the therapeutic application of inhaled CO should investigate itseffects in a larger population of patients, assessing the safety and optimaldosing schemes. Only when such studies show positive results, more severeindications as COPD exacerbations could be investigated with sufficientconfidence.

Improvements in treatment of COPD exacerbations in the near future.

In the upcoming years, treatment of COPD exacerbations should be improvedby various approaches. Both basic scientific research and clinical research isneeded to achieve this.

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Firstly, novel anti-inflammatory drugs should be developed for the treatment ofCOPD exacerbations. There is a need for novel drugs, since the effects ofsteroids in the treatment of exacerbations are modest. To make this possible,there is a need for models to test these. So far, there are no animal modelsresembling COPD exacerbations. The development of validated COPDexacerbation models in animals would be a step forward in the search for moreefficient treatments of COPD exacerbations.

Secondly, as long as we do not have more efficient therapies in the treatment ofCOPD exacerbations, we need to improve the current treatment. An importantstep in this is the assessment of the optimal dose, and duration of steroidtreatment. There is hardly any data on the optimal dose and duration of systemicsteroid treatment in COPD exacerbations. Only one trial has assessed thedifference between 2 week and 8 week steroid treatment (33). Shorter steroidschedules have to the best of our knowledge not been compared. Given thelarge number of COPD patients treated with steroids every year, this urgentlycalls for future investigations to provide evidence. There is also a markedpaucity of data regarding the dose of systemic steroids. Clinical guidelinesrecommend prednisone 40 mg orally once/day for 10 days in patients with anacute exacerbation of COPD (34). This was based on a panel consensusjudgment.

Thirdly, we need a better understanding of the course of COPD exacerbations.Future studies should further investigate the relation between cause ofexacerbation, inflammatory mechanisms and susceptibility to treatment drugs.Previous studies have shown that the various causes of COPD exacerbationsare associated with different inflammatory patterns. It is likely that the variouscauses of exacerbation also require specific treatment. For instance, futureinvestigations need to assess whether exacerbations caused by bacteria with aneutrophilic inflammation pattern respond to steroid treatment at all. Perhapsthese types of exacerbations should be treated with antibiotics andbronchodilators only. On the other hand, viral exacerbations with may inducewith eosinophilia might respond reasonably well to steroid treatment, and mightnot benefit from treatment with antibiotics. Exacerbations caused bymicro-aspiration might only respond to anti-reflux therapy. A split-up ofexacerbations by its cause, and investigation of cause-specific treatment effects,might lead to a more efficient treatment of COPD exacerbations.Research on COPD exacerbations and its treatment is still a starting field;however it is getting more and more attention in recent years. Much researcheffort is needed in the next years to improve both the understanding of COPDexacerbations and its treatment, but the gains will undoubtedly be large.

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References

(1) van der Valk P, Monninkhof E, van der Palen J, Zielhuis G, van Herwaarden C. Effect ofdiscontinuation of inhaled corticosteroids in patients with chronic obstructive pulmonarydisease: the COPE study. Am J Respir Crit Care Med 2002; 166(10):1358-1363.

(2) Wouters EF, Postma DS, Fokkens B, Hop WC, Prins J, Kuipers AF et al. Withdrawal offluticasone propionate from combined salmeterol/fluticasone treatment in patients withCOPD causes immediate and sustained disease deterioration: a randomised controlledtrial. Thorax 2005; 60(6):480-487.

(3) Seemungal TA, Donaldson GC, Bhowmik A, Jeffries DJ, Wedzicha JA. Time course andrecovery of exacerbations in patients with chronic obstructive pulmonary disease. Am JRespir Crit Care Med 2000; 161(5):1608-1613.




(7) Christ-Crain M, Jaccard-Stolz D, Bingisser R, Gencay MM, Huber PR, Tamm M et al.Effect of procalcitonin-guided treatment on antibiotic use and outcome in lowerrespiratory tract infections: cluster-randomised, single-blinded intervention trial. Lancet2004; 363(9409):600-607.


(9) Stolz D, Christ-Crain M, Bingisser R, Leuppi J, Miedinger D, Muller C et al. Antibiotictreatment of exacerbations of COPD: a randomized, controlled trial comparingprocalcitonin-guidance with standard therapy. Chest 2007; 131(1):9-19.

(10) Ball P. Epidemiology and treatment of chronic bronchitis and its exacerbations. Chest1995; 108(2 Suppl):43S-52S.



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(13) Harding SM, Richter JE, Guzzo MR, Schan CA, Alexander RW, Bradley LA. Asthma andgastroesophageal reflux: acid suppressive therapy improves asthma outcome. Am J Med1996; 100(4):395-405.

(14) Ricciardolo FL. Mechanisms of citric acid-induced bronchoconstriction. Am J Med 2001;111 Suppl 8A:18S-24S.

(15) Casanova C, Baudet JS, Valle Velasco M, Martin JM, Aguirre-Jaime A, de Torres JP etal. Increased gastro-oesophageal reflux disease in patients with severe COPD. EurRespir J 2004; 23(6):841-845.

(16) Mokhlesi B, Morris AL, Huang CF, Curcio AJ, Barrett TA, Kamp DW. Increasedprevalence of gastroesophageal reflux symptoms in patients with COPD. Chest 2001;119(4):1043-1048.

(17) Ducolone A, Vandevenne A, Jouin H, Grob JC, Coumaros D, Meyer C et al.Gastroesophageal reflux in patients with asthma and chronic bronchitis. Am Rev RespirDis 1987; 135(2):327-332.

(18) Locke GR, Talley NJ, Weaver AL, Zinsmeister AR. A new questionnaire forgastroesophageal reflux disease. Mayo Clin Proc 1994; 69(6):539-547.

(19) Rascon-Aguilar IE, Pamer M, Wludyka P, Cury J, Coultas D, Lambiase LR et al. Role ofgastroesophageal reflux symptoms in exacerbations of COPD. Chest 2006;130(4):1096-1101.

(20) O'Byrne PM, Bisgaard H, Godard PP, Pistolesi M, Palmqvist M, Zhu Y et al.Budesonide/formoterol combination therapy as both maintenance and reliever medicationin asthma. Am J Respir Crit Care Med 2005; 171(2):129-136.


(22) Balbi B, Bason C, Balleari E, Fiasella F, Pesci A, Ghio R et al. Increased bronchoalveolargranulocytes and granulocyte/macrophage colony-stimulating factor during exacerbationsof chronic bronchitis. Eur Respir J 1997; 10(4):846-850.


(24) Fujimoto K, Kubo K, Yamamoto H, Yamaguchi S, Matsuzawa Y. Eosinophilicinflammation in the airway is related to glucocorticoid reversibility in patients withpulmonary emphysema. Chest 1999; 115(3):697-702.


(26) Wilkinson TM, Hurst JR, Perera WR, Wilks M, Donaldson GC, Wedzicha JA. Effect ofinteractions between lower airway bacterial and rhinoviral infection in exacerbations ofCOPD. Chest 2006; 129(2):317-324.

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(28) Biernacki WA, Kharitonov SA, Barnes PJ. Increased leukotriene B4 and 8-isoprostane inexhaled breath condensate of patients with exacerbations of COPD. Thorax 2003;58(4):294-298.

(29) Shindo K, Hirai Y, Fukumura M, Koide K. Plasma levels of leukotriene E4 during clinicalcourse of chronic obstructive pulmonary disease. Prostaglandins Leukot Essent FattyAcids 1997; 56(3):213-217.

(30) Celik P, Sakar A, Havlucu Y, Yuksel H, Turkdogan P, Yorgancioglu A. Short-term effectsof montelukast in stable patients with moderate to severe COPD. Respir Med 2005;99(4):444-450.

(31) White AJ, Gompertz S, Bayley DL, Hill SL, O'Brien C, Unsal I et al. Resolution ofbronchial inflammation is related to bacterial eradication following treatment ofexacerbations of chronic bronchitis. Thorax 2003; 58(8):680-685.

(32) Lee M, Goodbourn S. Signalling from the cell surface to the nucleus. Essays Biochem2001; 37:71-85.



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Nederlandse samenvatting

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Nederlands samenvatting

Wat is COPD?

Chronic obstructive pulmonary disease (COPD) is een ziekte waarbij er eenblijvende vernauwing van de luchtwegen in de long optreedt, die voornamelijkveroorzaakt wordt door roken. Patiënten met COPD hebben een abnormaleontstekingsreactie in de longen door het roken van sigaretten en inhalatie vanschadelijke gassen. COPD bestaat uit 2 vormen: emfyseem (een verlies aanweefsel van de longblaasjes, waardoor “de rek uit de long” raakt en er mindergoede opname van zuurstof en afgifte van koolzuur mogelijk is) en chronischebronchitis (een chronische ontsteking aan de luchtwegen waarbij dagelijks slijmopgehoest wordt). De voornaamste klachten van COPD patiënten zijnkortademigheid bij inspanning, hoesten en overmatige slijmproductie van deluchtwegen. Sommige patiënten hebben frequent periodes met een verergeringvan klachten, exacerbaties genoemd. De behandeling van COPD bestaatvoornamelijk uit inhalatie van luchtwegverwijders, en bij patiënten die vaak eenexacerbatie hebben, worden ontstekingsremmers in inhalatie vorm (inhalatiesteroïden) voorgeschreven als dagelijkse behandeling.

De luchtwegen van patiënten met COPD zijn gevoelig voor virale of bacteriëleinfecties en luchtverontreiniging. Bij blootstelling aan deze zaken kan een min ofmeer plotselinge verergering van klachten onstaan: een exacerbatie. Hierbijtreedt een ontstekingsreacting op, die doorgaans wordt behandeld metontstekingsremmers in tablet vorm: een prednisolon stootkuur. Daarnaastworden deze exacerbaties behandeld met maximaal gebruik vanluchtwegverwijders. Exacerbaties vormen een belang aspect van COPDaangezien ze een negatieve invloed hebben op de kwaliteit van leven van depatiënt, en de levensverwachting van patiënten wordt bekort. Voor demaatschappij zijn de kosten van de behandeling van exacerbaties aanzienlijk,zeker als de patiënten in een ziekenhuis opgenomen moeten worden. Daarom ishet belangrijk deze exacerbaties te voorkomen, en als ze toch optreden ze danzo effectief mogelijk te behandelen.

De onderzoeksvraagstellingen in dit proefschrift

In dit proefschrift zijn de resultaten van 2 klinische studies beschreven. Deeerste is genaamd de Symbexco-studie, een onderzoek waarin werd bestudeerdof COPD exacerbaties met inhalator behandeld kunnen worden in plaats vanmet de prednisolon tabletten waar normaal mee wordt behandeld.Met behulp van de gegevens die we in dit onderzoek hebben verzameld hebbenwe de volgende onderwerpen onderzocht:

Patiënten met COPD krijgen vaak een dagelijkse behandeling metontstekingsremmers in inhalatievorm voorgeschreven. We

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onderzochten de vraag: wat gebeurt er met de ontsteking en klachtenvan de luchtwegen van patiënten met COPD, wanneer de dagelijkseonderhoudstherapie met ontstekingsremmers wordt gestopt (hoofdstuk3, hieronder samengevat onder “Gevolgen stoppen vanonderhoudstherapie met ontstekingsremmers)?Om informatie te krijgen over de ontsteking in de luchtwegen, vragen wedeelnemers verneveld zout te inhaleren, zodat ze gemakkelijk slijm ophoesten. Bij sommige patiënten treedt hierbij een vernauwing van deluchtwegen op. We onderzochten de vraag of het veilig is om patiëntenverneveld zout te laten inhaleren ook wanneer zij al een sterkereluchtwegvernauwing hebben tijdens een exacerbatie (hoofdstuk 4,hieronder samengevat onder “Veiligheid inhalatie verneveld zout”.Tijdens een verergering van luchtwegklachten bij patiënten met COPDwordt ook de ontsteking in de luchtwegen erger. We onderzochtenwelke kenmerken van de ontsteking verergeren, en of dit gerelateerd isaan de oorzaak van de exacerbatie (hoofdstuk 5, hieronder samengevatonder “Verandering van luchtwegontsteking bij een COPDexacerbatie”).We onderzochten de vraag of het mogelijk is een exacerbatie vanluchtwegklachten te behandelen met ontstekingsremmers per inhalatiein plaats van met prednisolon tabletten (ook wel stootkuur genoemd),waarvan bekend is dat er meer bijwerkingen zijn (hoofdstuk 6, hierondersamengevat onder: “Resultaten behandeling Symbexco studie”).

In de tweede studie werd onderzocht of het mogelijk is met een lage doseringingeademd koolmonoxide de ontsteking in de luchtwegen van patiënten metCOPD, die altijd in enige mate aanwezig is, geremd kan worden. De resultatenzijn beschreven in hoofdstuk 7, en samengevat onder resultaten koolmonoxidestudie

Gevolgen stoppen van onderhoudstherapie met ontstekingsremmers

Een gedeelte van patiënten met COPD gebruikt geïnhaleerdeontstekingsremmers als dagelijkse therapie. Volgens de richtlijnen wordt ditvoornamelijk voorgeschreven aan patiënten die regelmatig een exacerbatiehebben. In de dagelijkse praktijk zijn er ook patiënten met een minder ernstigCOPD, die ontstekingsremmers per inhalatie gebruiken. Deze patiënten zoudeneigenlijk dit medicijn niet meer moeten gebruiken. In de Symbexco-studievroegen we patiënten te stoppen met deze onderhoudstherapie en hebben wede verandering van de ontsteking, longfunctie en gezondheidsklachten gemeten.We zagen bij een deel van de patiënten dat de luchtwegklachten verergerden 2maanden na het stoppen van de onderhoudstherapie. We hebben onderzochtwelke veranderingen in ontsteking in de luchtwegen plaatsvonden, hoe dezetoename in ontsteking gerelateerd was aan een toename in klachten, en welkeontstekingskenmerken aan het begin voorspelden bij welke patiënten eenverergering in klachten op zou treden. We vonden dat het stoppen van deonderhoudstherapie leidde tot een toename van de luchtwegvernauwing, en een

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verslechtering van de kwaliteit van leven. De ontsteking in de luchtwegen namop allerlei manieren toe (verschillende cellen en ontstekingsstoffen). Detoename van één specifieke cel, de eosinofiel, een ontstekingscel die ook bijastma een grote rol speelt, was gerelateerd aan een toentame van deluchtwegvernauwing.

Veiligheid inhalatie verneveld zout

Om informatie over de ontsteking in de luchtwegen te verkrijgen vroegen wepatiënten met COPD verneveld zout te inhaleren, zodat ze gemakkelijk slijm opkunnen hoesten. Dat slijm wilden we namelijk onderzoeken om meer te weten tekomen over de ontstekingsreactie in de luchtwegen. Sommige patiënten werdenkortademig door het vernevelde zout; er trad een vernauwing van de luchtwegenop. Daarom hielden we tijdens deze procedure nauwgezet de longfunctie in degaten. Uit eerdere onderzoeken weten we dat in de stabiele fase van COPD hetveilig is verneveld zout te laten inhaleren, ook door patiënten met een ernstigeluchtwegvernauwing. Er was echter weinig beschreven over de veiligheid vaninhalatie van verneveld zout tijdens een COPD exacerbatie.

We vergeleken in welke mate de luchtwegen zich vernauwen door hetvernevelde zout tijdens een exacerbatie, en hoe dit zich verhield tot devernauwing van de luchtwegen in de stabiele fase van de ziekte. Verder kekenwe wat de kenmerken waren van de patiënten bij wie een sterkere/ernstigerevernauwing optreedt, en hoe we deze sterkere vernauwing op voorhand kunnenvoorspellen. We vonden dat tijdens exacerbatie een vergelijkbare vernauwingoptrad als die in de stabiele fase. Daarom adviseren we om bij patiënten bij wiein de stabiele fase een sterke luchtwegvernauwing optreedt tijdens inademingvan verneveld zout, extra voorzichtig te zijn door oplossingen te gebruiken meteen lagere zout concentratie en voorzichtig de zout concentraties op te hogen.

Verandering van luchtwegontsteking bij een COPD exacerbatie

Een COPD exacerbatie kan verscheidene oorzaken hebben: de belangrijkstezijn een virale ontsteking, een bacteriële ontsteking, blootstelling aanverontreinigde lucht, of een combinatie hiervan. Tijdens een exacerbatie treedtvaak ook een verergering van luchtweg ontsteking op. De verschillendeoorzaken van een exacerbatie bij patiënten met COPD veroorzakenverschillende patronen van ontsteking in de luchtwegen. We hebben onderzochthoe deze ontstekingspatronen er uitzien, en in het bijzonder wat voor eenontstekingspatroon een bacteriële verwekker veroorzaakt. Dit laatste kan voorpatiënten zeer belangrijk zijn, omdat je misschien uit het ontstekingspatroon opzou kunnen maken, of er met antibacteriële therapie moet worden gestart. Nukan er pas met zekerheid gezegd worden dat er een bacteriële oorzaak is,wanneer de bacteriën in het slijm gekweekt zijn, wat wel 5 dagen kan duren.

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We vonden dat alle soorten ontstekingscellen in hogere concentraties in hetslijm aanwezig zijn tijdens een exacerbatie vergeleken met een stabiele fase.Een bacteriële verwekker veroorzaakte naast de toename in ontsteking in hetslijm ook een toename in ontsteking in het bloed. We hebben onderzocht welkeontstekingswaarden in het slijm of in het bloed het best een bacterieleverwekker als oorzaak voorspelt. We vonden dat de concentratie van tumornecrosis factor-a in het slijm, een “boodschapper-eiwit” dat deontstekingsreactie op gang brengt, een goede voorspellende waarde had voorhet feit dat de oorzaak van de exacerbatie bacterieel was. Grotere onderzoekenzijn nodig, om precies vast te stellen welke waarde als afkappunt gebruikt moetworden om een bacteriële oorzaak zo zeker mogelijk te maken.

Resultaten proefbehandeling Symbexco studie:

Doel van de studie:

Het doel van de studie is te onderzoeken of het mogelijk is om een COPDexacerbatie te behandelen met een inhalator met daarin een combinatie vaneen langwerkende luchtwegverwijder en een ontstekingsremmer, in plaats vande prednisolon stootkuur die normaal gesproken bij een exacerbatie wordtgegeven. Het grote voordeel van behandelen met inhalatoren is dat demedicijnen rechtstreeks in de luchtwegen terecht komen. Daardoor komen zeminder in het bloed en de rest van het lichaam en hebben daar dan ook minderbijwerkingen. De deelnemers die een exacerbatie meldden zijn 2 weken langbehandeld met ofwel A: inhalator met hierin een luchtwegverwijder enontstekingsremmer, ofwel B: prednisolon stootkuur, ofwel C: placebo. In totaalzijn 45 patiënten behandeld voor een exacerbatie. Zowel de deelnemendepatiënten als de behandelaars en onderzoekers wisten daarbij niet wie welkebehandeling kreeg.

We vergeleken in eerste instantie de resultaten van de behandeling met deinhalatoren met hierin een luchtwegverwijder en ontstekingsremmer versusbehandeling met placebo. Dit deden we om te kijken of de behandeling metinhalatoren met hierin een luchtwegverwijder en ontstekingsremmer deontsteking die samen gaat met een exacerbatie verbetert.Vervolgens vergeleken we de resultaten van de behandeling met inhalator methierin een luchtwegverwijder en ontstekingsremmer versus behandeling met deprednisolon stootkuur (de gebruikelijke behandeling). Dit deden we om eenindruk te krijgen of de behandeling met inhalator met hierin eenluchtwegverwijder en ontstekingsremmer een verbetering van de ontstekinggeeft die vergelijkbaar is met de verbetering bij de gebruikelijke behandeling metde prednisolonkuur, en om te bezien of de behandeling met de inhalator methierin een luchtwegverwijder en ontstekingsremmer minder bijwerkingen had.

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Resultaten van de behandelingen op de ontsteking

We hebben de deelnemers die behandeld zijn voor een verergering vanklachten onder andere op de eerste dag en na 2 weken gevraagd eenzoutoplossing in te ademen, zodat ze makkelijker slijm op hoesten. In dit slijmhebben we ontstekingscellen gemeten.

De ontstekingscel, die de belangrijkste rol speelt tijdens een verergering vanklachten, is de eosinofiel. De volgende veranderingen traden op door debehandelingen op het aantal eosinofielen in het slijm:

Behandeling met inhalator met hierin een luchtwegverwijder enontstekingsremmer gaf een afname van eosinofielen in het slijm van 57%.Behandeling met prednisolon stootkuur gaf een afname van eosinofielen inhet slijm van 58%.Behandeling met placebo (nepmedicijn) gaf een toename van eosinofielenin het slijm van 24 %.

De afname van het aantal eosinofielen door zowel de inhalers als deprednisolonstootkuur was groter dan je op basis van toeval zou kunnenverwachten. Met andere woorden: dat effect moet echt aan het actieve medicijnliggen. In statistisch jargon: het gemeten effect berust niet op toeval.

Resultaten van de behandelingen op de longfunctie

Bij de deelnemers die behandeld zijn voor hun exacerbatie hebben we tijdens debehandeling longfunctie gemeten.We vonden dat bij de deelnemers die met de inhalatoren met hierin eenluchtwegverwijder en ontstekingsremmer waren behandeld, een grotereverbetering optrad in de vernauwing van de luchtwegen, dan bij deelnemers diemet de prednisolonstootkuur of met placebo waren behandeld. Maar detoename was variabel tussen deelnemers zodat we niet met zekerheid kunnenzeggen dat de toename groter was dan je op basis van toeval zou kunnenverwachten. Om te kijken of deze verbetering van de vernauwing van deluchtwegen wel echt is (niet op toeval berust), zou je in de toekomst dit metmeer deelnemers moeten onderzoeken.

Resultaten van de behandeling op de gezondheids vragenlijsten enklachten

We hadden de deelnemers aan de studie tijdens de behandeling van deexacerbatie uitgenodigd vragenlijsten over de gezondheidstoestand in te vullen.

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We vonden dat de behandeling van de exacerbatie met inhalator met eenluchtwegverwijder en ontstekingsremmer de grootste vooruitgang op het gebiedvan de gezondheidstoestand gaf.

Tijdens de behandelingsperiode hebben de deelnemers een dagboekbijgehouden, en hierin de ernst van hun klachten aangegeven. We vonden datzowel tijdens de behandeling met de inhalator met een luchtwegverwijder enontstekingsremmer als met de prednisolonstootkuur een verbetering optrad inde klachten vergeleken met tijdens de placebobehandeling.

Effecten van de behandeling op bijwerkingen in het lichaam

In het bloed konden we meten in hoeverre er bijwerkingen optreden door debehandeling met ontstekingsremmers. De maat die we hiervoor gebruikten iscortisol. Cortisol kan worden onderdrukt door ontstekingsremmers, waardoorbijwerkingen als onder andere botontkalking kunnen ontstaan. Dit kan optredenals patiënten veel prednisolon nodig hebben.We vonden dat de prednisolon stootkuur inderdaad het cortisol onderdrukte. Bijde behandeling met de inhalator met een luchtwegverwijder enontstekingsremmer was dit niet het geval.

Slotconclusie van de Symbexco-studie

Behandeling van een exacerbatie van COPD met inhalers met eenluchtwegverwijder en een ontstekingsremmer is goed mogelijk. Het geeft eenverbetering van de ontsteking, de klachten en de gezondheidstoestandvergeleken met behandeling met een nepmedicijn. We konden uit de studie nietopmaken of behandeling met inhalator met een luchtwegverwijder enontstekingsremmer beter of slechter of even goed werkt als behandeling met degebruikelijke prednisolon stootkuur. Wel zagen we dat de behandeling met deprednisolon stootkuur in het lichaam bijwerkingen geeft, die we niet vonden bijbehandeling met inhalator met een luchtwegverwijder en ontstekingsremmer.

Samenvatting resultaten koolmonoxidestudie

Doel van de studie

Het doel van de koolmonoxidestudie was te onderzoeken of een lage doseringingeademd koolmonoxide een ontstekingsremmend effect heeft in deluchtwegen van patiënten met COPD. Koolmonoxide is algemeen bekend alshet geurloze gas dat -ingeademd in hoge concentraties- dodelijk kan zijn. Er zijnechter ook gunstige effecten van koolmonoxide. In cel- en diermodelstudies is

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gebleken dat koolmonoxide sterke ontstekingsremmende effecten heeft. Dit zoudus gunstig zijn in COPD, omdat daarbij een verhoogde ontstekingsreactieontstaat. De luchtwegen maken zelf ook actief koolmonoxide aan op hetmoment dat de ontsteking in de luchtwegen verergert. Overigens zit er ook insigarettenrook koolmonoxide.In hoge concentraties is koolmonoxide giftig. We hadden onze concentratie zogekozen dat deze vergelijkbaar is met de concentratie die rokers doorsigarettenrook binnen krijgen, wanneer ze 1 pak sigaretten per dag zoudenroken. Deze concentratie blijft ver onder de giftige concentraties.

Om te onderzoeken of ingeademd koolmonoxide inderdaad een remmendewerking heeft op de ontsteking van de luchtwegen bij met COPD, hebben weeen onderzoek opgezet waarbij we deelnemers vroegen in 2 periodes van 4dagen achtereen gedurende 2 uur per dag een lage concentratie koolmonoxide,of gewone lucht uit een gascilinder in te ademen. Deze periodes waren inwillekeurige volgorde, zonder dat de onderzoekers of de deelnemers wisten inwelke periode welk gas geïnhaleerd werd. Op de 5de dag van beide periodeshebben we de effecten van de behandeling gemeten.

Het belangrijkste waar we in het onderzoek naar keken was de ontsteking in hetopgehoeste slijm. We vonden dat de ontsteking door de ingeademdekoolmonoxide over het algemeen verbeterde, maar de resultaten waren tevariabel tussen de proefpersonen om de uitslag met voldoende stelligheid tekunnen presenteren. Studies met grotere groepen deelnemers zullen moetenbevestigen of koolmonoxide de ontsteking in een zodanige mate verbetert datpatiënten het verschil ook echt merken en de behandeling met ingeademdkoolmonoxide een reëele mogelijkheid wordt. We zagen dat voornamelijk eenontstekingscel die in astma een rol speelt, de al eerder genoemde eosinofiel,verbeterde door de koolmonoxide. Daarom zijn we in het UMCG nu bezig eenonderzoek op te zetten, waarin de ontstekingsremmende effecten vankoolmonoxide bij astma patiënten wordt onderzocht.

Daarnaast hebben we onderzocht of de longfunctie en de kwaliteit van levenverbeterde door de koolmonoxide. Dit was niet het geval. Misschien dat tijdenseen exacerbatie, waarbij de luchtwegontsteking een grotere rol speelt,koolmonoxide hier wel een effect op zal blijken te hebben. Ook dit zal in detoekomst onderzocht mochten worden.

Algemene samenvatting

Samenvattend werd in dit proefschrift uitgebreid gekeken naar deontstekingsreactie in de luchtwegen van mensen met COPD. Deze ontsteking iserg belangrijk bij het ontstaan, en bij de geleidelijke achteruitgang van COPD. Bijeen exacerbatie van COPD is de ontstekingsreactie nog weer heftiger, enafhankelijk van het uitlokkende moment, ook anders van aard. Tot nu toe is het

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steeds erg moeilijk gebleken deze ontstekingsreactie goed te behandelen metmethodes met aanvaardbare niveaus van bijwerkingen. In dit proefschriftworden twee nieuwe methodes geïntroduceerd waar we hoopvolle resultatenmee boekten en die nu in grotere en meeromvattende studies uitgetest zullengaan worden.

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Dankwoord

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Dankwoord

Heel erg bedankt iedereen!

In de eerste plaats zou ik alle patiënten willen bedanken die vrijwillig aan destudies mee hebben gedaan. Uw deelname heeft er toe geleid dat we veelinformatie hebben verkregen over de luchtweginflammatie tijdens COPDexacerbaties, en de effecten van de behandeling hierop. Zonder uw geweldigeinzet was dit proefschrift niet mogelijk geweest!

Verder zou ik alle mensen willen bedanken die aan de studies hebbenmeegewerkt, en in het bijzonder:

Mijn promotor Prof dr H.A.M. Kerstjens,Beste Huib, ik heb zeer veel geleerd van je deskundigheid en kritischebenadering bij het opzetten en uitvoeren van onderzoek. Je enthousiasme voorwetenschappelijk onderzoek werkte zeer stimulerend, en je motiveerde mijdaarmee een stage in het buitenland te ondernemen, waar ik je zeer dankbaarvoor ben. Hartelijk dank voor al je inspanningen de afgelopen jaren!

Mijn promotor Prof dr D.S. Postma,Beste Dirkje, ik heb grote bewondering voor je werktempo en doortastendheid:een mail of telefoontje aan je, en het is geregeld. Ik heb door jou altijd hetvertrouwen gehad dat mijn promotietraject zou slagen, je straalde uit “eenlevend vangnet voor onderzoekers” te zijn. Je hebt veel aandacht besteed aanmijn ontwikkeling, en me veel steun gegeven, in het bijzonder tijdens deafronding van het proefschrift. Ik ben je hier zeer dankbaar voor!

Mijn promotor Prof. dr G.H. KoëterBeste Gerard, je toonde een geweldige interesse voor de mensen die op deafdeling werkten, en hierdoor creëerde je een groot vertrouwen. Iedereen konmet problemen bij je terecht, en je nam hier uitgebreid de tijd voor. De laatstebesprekingen bij je thuis op de koffie heb ik als erg gezellig ervaren. Het is mijeen grote eer als laatste onderzoeker bij je te mogen promoveren!

Prof dr A.J.M. van Oosterhout, prof dr J.W.J. Lammers, and prof W. Macnee,many thanks for your willingness to be a member of the review committee of thisthesis!

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Jeroen,bedankt voor al je werk aan de Symbexco studie, het is een enorme klusgeweest!

DJ TNX!!!

Martin Boorsma,bedankt voor, je hulp, adviezen en het monitoren van de trial.

De longfunctie afdeling,bedankt voor al jullie hulp bij de trials, ik heb genoten van de gezellige enontspannen sfeer op jullie afdeling!

Het lab Allergologie en Longziekten,Antoon, Henk K, Sicco, Ibolya, Dorothea en Brigitte, dank voor al jullie hulp bijhet opzetten van de studies en de analyses van de samples!

Secretariaat longziekten,Trudy en Evelyn dank voor al jullie werk!

Alle longartsen en arts-assistenten van de afdeling longziekten: dank voor deprima samenwerking en werksfeer! Ik hoop jullie in de toekomst als collegasnog vaak tegen te komen.

Judith, Marike en Jan, dank voor jullie hulp bij de analyses en hetrandomisatie-proces van de trials!

Alle GRIAC-leden, dank voor het waardevolle commentaar en de stimulerendediscussies tijdens de research besprekingen!

Dear Bill, Ellen, Rodger, and Eileen, many thanks for your hospitality, Edinburghwas a second home for me! Paul, Birgit, Craig, Senlin, Roberto, Carl, Irene, Sue,John, Joy, and David, it is a miracle that no one got killed; medical doctors in thelab are always a liability! Many thanks for the briljant time!

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Mijn kamergenoten en collega-onderzoekers,Judith, Renske, Wouter, Sandra, Fransien, Franke, Jos, Maarten, Henk, Toby,Hajo, Hester, Margot, Naomi, Jaap, Elisabeth, Brigitte, Siebrig, Juliet enJanWillem dank voor alle hulp en gezelligheid!

Mijn oud-huisgenoten,Namreh, Edwin, Bernardo en Dirk, dank voor de geweldige tijd! Ik mis devis-met wijn-avondjes en het pokeren! Thomas, Stephan, and Chris it was morethan a pleasure to be roommates!

Mijn paranimfen,Mirjam, Pep, ik ben blij dat jullie aan mijn zijde zullen staan tijdens deverdediging van dit proefschrift!

Harry en Marleen,Dank voor jullie betrokkenheid en het kritisch nakijken van de Nederlandsesamenvatting!

Mijn ouders en zusje,Bert, Femmie, Annamiek jullie zijn er altijd en onvoorwaardelijk voor me!

Lieve Heske,Dank je voor al je hulp en liefde. Dikke kus!

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university of groningen copd exacerbations, …in the 2005 update of the global initiative for...

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