NON-SPECIFIC INFLAMMATION DURING ACUTE EXACERBATION OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Mindaugas Vaitkus

NON-SPECIFIC INFLAMMATION

DURING ACUTE EXACERBATION

OF CHRONIC OBSTRUCTIVE

PULMONARY DISEASE

Summary of Doctoral Dissertation Biomedical Sciences, Medicine

(06B)

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The dissertation was prepared during 2009–201 at the Department of Pulmonology and Immunology of Medical Academy of Lithuanian University of Health Sciences.

Scientific supervisior

Prof. Dr. Raimundas Sakalauskas (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06B)

The dissertation is defended at the Medical Research Council of the Medical Academy of Lithuanian University of Health Sciences:

Chairman

Prof. Dr. Limas Kupčinskas (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06 B)

Members:

Prof. Dr. Asta Baranauskaitė (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06B)

Prof. Dr. Skaidrius Miliauskas (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06B)

Prof. Dr. Janina Didžiapetrienė (Vilnius University, Biomedical Sciences, Me- dicine – 06B)

Prof. Dr. Saulius Šatkauskas (Vytautas Magnus University, Biomedical Scien- ces, Biology – 01B)

Opponents:

Prof. Dr. Ramune Morkūnienė (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Biology – 01B)

Prof. Dr. Jolanta Dadonienė (Vilnius University, Biomedical Sciences, Medi- cine – 06B)

The dissertation will be defended in the open session of the Medical Research Council of Medical Academy of Lithuanian University of Health Sciences on the 26th of August, 2014 at 11 o’clock in the Large Auditorium of the Hospital of Lithuanian University of Health Sciences Kauno Klinikos.

Address: Eivenių 2, LT-50009 Kaunas, Lithuania.

The summary of the dissertation has been sent on the 24th of July, 2014.

The full text of the doctoral dissertation is available at the Library of Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Mindaugas Vaitkus

NESPECIFINIS UŽDEGIMAS

PA

ŪMĖJUS LĖTINEI

OBSTRUKCINEI PLAU

ČIŲ LIGAI

Daktaro disertacijos santrauka Biomedicinos mokslai,

medicina (06B)

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Disertacija rengta 2009–2014 metais Lietuvos sveikatos mokslų universiteto Medi- cinos akademijos Pulmonologijos ir imunologijos klinikoje.

Mokslinis vadovas

Prof. dr. Raimundas Sakalauskas (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, medicina – 06B)

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademi- jos Medicinos mokslo krypties taryboje:

Pirmininkas

prof. habil. dr. Limas Kupčinskas (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, medicina – 06B).

Nariai:

prof. dr. Asta Baranauskaitė (Lietuvos sveikatos mokslų universitetas, Medi- cinos akademija, biomedicinos mokslai, medicina – 06B);

prof. dr. Skaidrius Miliauskas (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, medicina – 06B);

prof. dr. Janina Didžiapetrienė (Vilniaus universitetas, biomedicinos mokslai, medicina – 06B);

prof. dr. Saulius Šatkauskas (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija – 01B).

Oponentai:

prof. dr. Ramunė Morkūnienė (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, biologija – 01B);

prof. dr. Jolanta Dadonienė (Vilniaus universitetas, biomedicinos mokslai, medicina – 06B).

Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Medicinos akademijos medicinos mokslo krypties tarybos posėdyje 2014 m. rugpjūčio 26 d. 11 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikų Didžiojoje auditorijoje.

Adresas: Eivenių g. 4, LT-50009 Kaunas, Lietuva. Disertacijos santrauka išsiųsta 2014 m. liepos 24 d.

Disertaciją galima peržiūrėti Lietuvos sveikatos mokslų universiteto bibliotekoje. Adresas: Eivenių g. 6, LT-50162 Kaunas, Lietuva.

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ABBREVATIONS

AECOPD Acute exacerbation of Chronic obstructive pulmonary disease BMI Body mass index

COPD Chronic obstructive pulmonary disease CRB C reaktyve protein

DHR-123 Dihydrorodamine-123 DTT Dithiothreitol

EDTA Ethylene diamine tetra-acetic acid ELISA Enzyme-linked immunosorbent assay FEV1 Forced expiratory volume in 1 sec.

FVC Forced vital capacity

GOLD Global Initiative for Chronic Obstructive Lung Disease IL Interleukin

PMA Phorbol 12-myristate 13-acetate

rCOPD Remission of Chronic obstructive pulmonary disease ROS Reactive oxygen species

S. aureus Staphylococcus aureus

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1. INTRODUCTION

Chronic Obstructive Pulmonary Disease (COPD) is characterized by airflow limitation which is not fully reversible. It is a treatable and preventable disease but progressive and associated with an abnormal inflammatory response of the lung to noxious particles or gases. The causes of COPD are multifactor involving genetic and environmental factors. Moreover, smoking is a major environmental risk factor for COPD. Morbidity and mortality among patients with COPD are for a large part related to acute exacerbations (AECOPD), which occur one to three times a year. Studies have shown that inflammation in COPD are co- dependent and strongly interrelated processes not only in local, but also in systemic inflammation in the lungs. The processes of inflammation in stable COPD have been extensively investigated. The most common cause of AECOPD is infections in the airways [5]. Some studies have shown that at least 50% of patients during AECOPD have bacteria in the lower respiratory tract. Various inflammatory cells participate in COPD mecha- nism and release different mediators (cytokines, chemokines, complement system, etc.) which play a key role in pathogenesis of the disease.

It is found that significantly increase count of inflammatory cells during AECOPD. Inflammatory cells produce higher amount of various inflammatory mediators in the airways. These mediators are expressed in the airways and in peripheral blood and change local and systemic inflammation.

Interleukin-8 is an important chemokine and chemoatractant of peripheral blood neutrophils. Peripheral blood and airway neutrophils, alveolar macrophages, epithelial cells produced and release increased amount of IL-8 during AECOPD.

It was shown that change activity of inflammatory cells during AECOPD compare with stable disease. But is unclear how change cellular activity during AECOPD depending on infection agent.

Thus, analysis of induced sputum neutrophils and macrophages and peripheral blood neutrophils and monocytes functional features would help understand the cellular features of local and systemic inflammation during AECOPD dependent on caused agent.

The aim of this study was to evaluate the non-specific inflammation

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The objectives of the study:

1. To investigate cellular composition of peripheral blood and induced sputum and evaluate possible relationship with lung function tests in patients during bacterial and non-bacterial acute exacerbation of chronic obstructive pulmonary disease.

2. To analyze induced sputum neutrophil and macrophage apoptosis, phagocytosis and the production of reactive oxygen species during bacterial and non-bacterial acute exacerbation of chronic obstructive pulmonary disease.

3. To evaluate peripheral blood neutrophil and monocyte apoptosis, chemotaxis, as well as peripheral blood neutrophil phagocytosis and the production of reactive oxygen species during bacterial and non- bacterial acute exacerbation of chronic obstructive pulmonary disease. 4. To investigate IL-8 concentration in peripheral blood and induced

sputum, CRB concentration in peripheral blood and possible relationship with cellular activity and lung function tests during acute bacterial and non-bacterial acute exacerbation of chronic obstructive pulmonary disease.

Scientific novelty of the study

At the beginning of this study only the results of local and systemic immune response during stable COPD were published. To author’s knowledge the investigation of the cellular activity during AECOPD and then after recovery in the same patients group were published for the first time. An unknown difference of induced sputum neutrophil and macrophages apoptosis, phagocytosis and ROS production in bacterial and non- bacterial AECOPD was also analyzed. The study explored more important information about the cellular activity of COPD pathogenesis.

2. MATERIALS AND METHODS

The Kaunas Regional Ethics Committee for Biomedical Research approved the study, and each participant gave written informed consent.

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2.1. Study population and design

Fifty patients with COPD during acute exacerbations and the same 50 patients after recovery from exacerbation were enrolled to this study. COPD was diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease criteria. The inclusion criteria were as follows: defined exacerbation of COPD according to the criteria modified by Annthonisen et al., postbronchodilator FEV1/FVC, <0.70; FEV1, <80% of

predicted; β2-agonist reversibility, less than 15% and/or 200 mL; smoking

history, more than 10 pack/years; and no history of asthma, bronchiectasis, carcinoma of the bronchus, or other significant respiratory disease. All COPD patients had not been treated with systemic steroids and/or antibiotics for at least 1 month before the study.

Two control groups were recruited for the study: 10 healthy indi- viduals with a smoking history of more than 10 pack/years and 10 healthy nonsmokers with a normal pulmonary function.

Smoking and drug history, sex, body mass index (BMI), comorbid conditions, and findings of St. George Respiratory Questionnaire and pulmonary function test were recorded during exacerbation. Peripheral blood and sputum samples were taken for further investigations. To calculate pack-years of smoking, the total number of years of smoking was multiplied by the average number of cigarettes smoked per day and divided by 20 (Number of pack-years = (number of cigarettes smoked per day × number of years smoked)/20 (1 pack has 20 cigarettes)). BMI was calculated as weight in kilograms divided by the square of height in meters.

The patients during AECOPD were treated with oral steroids (pred- nisolone, 40 mg) for 10 days, and continued to use bronchodilators; antibiotics were prescribed if bacteria from sputum were isolated. It was considered that patients recovered from exacerbations, i.e., became stable, within a period of 35 days. The St. George Respiratory Questionnaire was administered repeatedly, and peripheral blood and sputum samples were taken as well.

The patients with AECOPD were divided in two groups as bacterial and non-bacterial AECOPD depending on isolating of bacteria from induced sputum.

The study consisted of:

• An investigation of cellular composition in peripheral blood and induced sputum;

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• Analysis of peripheral blood (neutrophil, monocyte) and induced sputum (neutrophil, macrophage) cellular activity: apoptosis, chemotaxis, phagocytosis, production of ROS;

• IL-8 concentration in peripheral blood and induced sputum; • CRB concentration in peripheral blood.

2.2. Lung Function

Pulmonary function was tested using a pneumotachometric spirometer "CustovitM" (Custo Med, Germany) with subjects in the sitting position, and the highest value of forced expiratory volume in 1 second (FEV1) and

forced vital capacity (FVC) from at least two technically satisfactory maneuvers differing by less than 5% was recorded. The results were compared with the predicted values matched for age and sex according to the standard methodology [15]. The subjects had not used short-acting β2-

agonists at least 8 hours, long-acting β2-agonists 12 hours, and long-acting

anticholinergics 24 hours before the test.

2.3. Peripheral blood collection and isolation of neutrophil and monocyte

Peripheral blood for the isolation of neutrophils and eosinophils were collected into sterile vacutainers with anti-coagulated EDTA. Peripheral blood neutrophils were isolated by high-density gradient centrifugation. The whole blood was layered on Ficoll-Paque PLUS (GE Healthcare, Finland) and centrifuged at 1000g for 30 minutes at room temperature. Peripheral blood neutrophil population was separated by hypotonic lysis of erythrocytes. Peripheral blood monocytes from all mononuclear cells were separated using magnetic separation (Monocyte isolation kit II, human; Miltenyi Biotec, USA). Isolated peripheral blood neutrophils and monocytes were diluted in a cell culture RPMI 1640 medium (Biological In-

dustries, Israel) at a final concentration of 2×106/mL. The viability of peripheral blood neutrophils and monocytes was checked by flow cytometry, and it always was > 95%.

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2.4. Sputum induction, collection and isolation of sputum neutrophils and macrophages

After the baseline FEV1 measurement, salbutamol (200 µg) was given

by inhalation, and the subjects inhaled 10 mL of sterile hypertonic saline solution (3%, 4%, or 5% NaCl) from an ultrasonic nebulizer (DeVilbiss Health Care, USA) at room temperature. The duration of each inhalation was 7 minutes and was stopped earlier after expectoration an adequate amount of sputum. After each period of inhalation, FEV1 was measured for

patient safety. Sputum was poured into a Petri dish and separated from saliva. A volume of 1x PBS equal to 8 times the weight of the sputum plugs was added and centrifuged at 790g for 15 minutes. The removed supernatant was vacuumed into sterile cold-resistant Eppendorf tubes and stored at –70°C for further ELISA analysis. A fourfold volume of freshly prepared 0.1% dithiothreitol (DTT; Sigma-Aldrich, Germany) was added to the cell pellets. The mixture was vortexed and placed on a bench rocker for 15 minutes at room temperature. Next, an equal volume of phosphate- buffered saline (PBS; Sigma-Aldrich, Germany) solution was added to DTT. The cell pellet was separated using a cell strainer (40 μm) (Becton Dickinson, USA). The mixture was centrifuged for 10 minutes at 4°C; the supernatant was aspirated. Total cell counts, percentage of epithelial cells, and cell viability were investigated using a Neubauer hemocytometer (Heinz-Herenz; Germany) by a microscope (B5 Professional, Motic, China), employing trypan blue exclusion method. Neutrophils for in vitro investigation from other cells were isolated by washing with PBS and macrophages enriched using magnetic isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, USA).

2.5. Serum Processing

Peripheral blood was collected into sterile tubes without additives (2×5 mL) and stored at room temperature for the surface clot formation (about 30 minutes). Then tubes were centrifuged at 1000g for 10 minutes at room temperature. In the upper layer of the sample, serum was vacuumed into sterile cold-resistant Eppendorf tubes and stored at –70°C for further ELISA analysis.

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2.6. Interleukin-8 Measurements

IL-8 levels in serum and induced sputum supernatants were determined by an enzyme-linked immunosorbent assay (ELISA) using a com- mercial IL-8 ELISA kit (IBL International, USA) according to the manufacturer’s instructions. The minimum detectable dose was 1.6 pg/ml and was defined as the IL-8 concentration corresponding to the average optical density of 20 replicates of the zero standard + 2 standard deviations.

2.7. C Reactive Protein Measurements

Serum CRP levels were measured using turbodimetric method using Synchron automatic system Unicel DxC 800 (Becman Coulter, USA).

2.8. Microbiological Examination

Microbiological sputum examination was performed using the standard methods in the Laboratory of Microbiology. AECOPD was defined as bacterial if patient have purulent sputum and (or) isolated bacteria from induced sputum [16-18].

2.9. Flow cytometric analysis for cellular activity

Flow cytometric measurements were performed with a FACSCalibur cytometer (Becton Dickinson, USA). For each determination, 104 events were acquired. The characteristic properties of peripheral blood neutrophils, monocytes, induced sputum neutrophils and macrophages (size and granularity/complexity) served to determine the purity of cellular suspensions and to monitor morphological changes after incubations and stimulations with tested substances. The total number of viable cells were quantified and identified by addition of propidium iodide (2 mg/mL, Calbiochem, Germany), which stains deoxyribonucleic acid of dead cells. Data were analyzed with the software CellQuest.

2.10. Apoptosis Assay

Isolated cells were resuspended in the annexin-binding buffer (pH 7.4) containing 50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2 (Invitrogen, USA) and incubated with fluorescein isothiocyanate-labeled (FITC)-

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annexin V (Invitrogen, USA) and propidium iodide (PI) for 15 minutes at room temperature in the dark. After the incubation, apoptosis was analyzed by flow cytometry using the CellQuest software (BD Biosciences, USA). Apoptotic cells were quantified as the percentage of the total population that was positive for FITC, but negative for PI. Necrotic cells were positive for both FITC and PI.

2.11. Chemotaxis

Chemotaxis in vitro was performed in a ten-well cell transmigration chamber (Neuro Probe, USA). The lower and upper wells of chamber were isolated by a polyvinylpyrrolidone-treated polycarbonate track-etch memb- rane, containing 2×106 3 μm/mm2 pores (Neuro Probe). The lower wells were preﬁlled with isotonic Percoll (GE Healthcare) and chemotactic factors – IL-8 at different concentrations (10, 30, or 100 ng/ml); C5a at different concentrations (10, 30 or 100 ng/ml). RPMI 1640 was used as a negative control. The upper wells were ﬁlled with cell culture (neutrophils, monocytes) suspension (2×106 /ml) and incubated for 2 h (37°C, 5% CO2).

After the incubation, the suspensions of upper and lower wells were resuspended in tubes for ﬂow cytometry. Non-migrated neutrophils or monocytes remained in the upper wells. The migration rate was calculated from the total number of the cells harvested from the lower well and expressed as percentage of the total input of neutrophils or monocytes into the upper compartment of the well. The number of migrated cells was calculated by ﬂow cytometry using liquid counting beads (BD Bioscien- ces, USA), according to the manufacturer’s recommendations. The amount of migrated neutrophils or monocytes was expressed in percentages.

2.12. Phagocytosis

Phagocytic activity of peripheral blood neutrophils and induced sputum neutrophils and macrophages in vitro was determined in sterile 96- well microplates (Falcon, BD, USA). To evaluate phagocytic capacity, neutrophils were stimulated with ﬂuorescein isothiocyanate (FITC)-labeled Staphylococcus aureus bacteria. Cells were stimulated with ratios of 0.7, 2, 6, 18, 56, and 167 FITC-labeled S. aureus bacteria per cell. To evaluate phagocytic capacity, neutrophil and macrophage cultures were added at a concentration of 2×106/ml and incubated for 2 h (37°C, 5% CO2). Cell

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mean ﬂuorescence intensity in neutrophil population (excitation wavelength, 488 nm).

2.13. Production of reactive oxygen species

ROS production in peripheral blood neutrophils and induced sputum neutrophils and macrophages was induced by chemical (phorbol 12-myristate 13-acetate [PMA], 0.1, 0.3, 1, 3, 10, and 30 nM) and biological (0.7, 2, 6, 18, 56, and 167 S. aureus bacteria per cell) factors in sterile 96-well microplates (Falcon, BD, USA). For the detection of generated ROS, a nonfluorescent dye, dihydrorhodamine 123 (DHR-123; final concentration, 750 ng/mL; Invitrogen, USA), was added. DHR-123 by interaction with intracellular ROS is oxidized to green-fluorescent rhodamine 123 (cata- lyzed by cellular myeloperoxidase). The plates were filled with ROS- stimulating factors, coated with cell cultures, and incubated for 45 minutes (with PMA) or 2 hours (with S. aureus at 37°C, 5% CO2). The relative

amount of generated ROS was measured by flow cytometry determining the median fluorescence intensity (MFI) in the cell population (excitation wavelength, 488 nm).

2.14. Mathematical – statistical data analysis

Statistical analysis of the study data was performed using GraphPad

Prism 6 for Windows software package. The power of completed expe-

riments was calculated using program GraphPad StatMate 2. Acceptable power was choosing more than 0.8. The differences between two inde- pendent study groups were evaluated using Mann-Whitney test, to compare two dependent study groups – Wilcoxon parameter. Differences between more than two independent study groups were calculated using Kruskal-

Wallis test. Correlation was evaluated using Spearman correlation coef- ficient. Statistical significance was assumed at p<0.05.

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3. RESULTS

3.1. Characteristics of Study Population

The demographic and clinical characteristics of the study population are summarized in Table 3.1.1. All the patients (n=50) had AECOPD 2 days before the study. There was no significant difference in age, BMI, and smoking history comparing the rCOPD (remission of COPD) group with “healthy” smokers and non-smokers. FEV1 and FVC were signifi-

cantly lower in the bacterial AECOPD group than in the rCOPD after bacterial AECOPD group as well as in the non-bacterial AECOPD and rCOPD after non-bacterial AECOPD groups (p<0.05). The SGRQ score was significantly greater in the patients with AECOPD and rCOPD than in the “healthy” smokers and non-smokers (p<0.05).

Table 3.1.1. The characteristics of the study population

Variable Bacterial AECOPD (n=23) Non- bacterial AECOPD (n=27) Bacterial rCOPD (n=23) Non- bacterial rCOPD (n=27) „Healthy“ smokers (n=10) Healthy Non- smokers (n=10) Age, years 66±3,1 65,6±2,8 60,3±3,2 61,0±14 BMI, kg/m2 23,9±2,0 23,0±1,4 26,4±1,5 25,7±1,7 FEV1, L 1,20±0,2* 1,34±0,1 1,22±0,1** 1,36±0,1 4,38±0,2# 4,50±0,1# FEV₁, % of pred. 33,8±3,3* 41,4±3,0 36,1±11,2** 42,0±3,9 # 112,3±12,7 112,7±7,8# FVC, L 2,20±0,1* 2,35±0,1 2,21±0,1** 2,36±0,1 5,27±0,2# 5,26±0,2# FEV₁/FVC 56,6±9,0* 56,8±3,3 55,2±5,0** 57,5±3,0 83,2±4,8# 85,7±4,1# Smoking history, pack-years 33,2±5,1 29,7±6,8 24±3,1 – SGRQ, score 53,4±2,5* 44,4±2,5 56,3±1,7** 47,7±3,0 # 27,8±3,7 22,6±2,7 #

Data are expressed as mean±standard deviation. AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD. FEV1 – forced expiratory volume in one

second; FVC – forced vital capacity; BMI – body mass index; SGRQ – St. George Respiratory Questionnaire; NA – not applicable.

*p<0.05 compared with bacterial rCOPD; **p<0,05, compared with non-bacterial rCOPD; #p<0,05, compare with all COPD groups.

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3.2. Cellular composition in peripheral blood and induced sputum

Induced sputum neutrophils were predominant cell type in induced sputum of patients with bacterial AECOPD compared with non-bacterial AECOPD and remission after bacterial AECOPD (p<0.05). The amount of induced sputum neutrophils was higher during non-bacterial AECOPD than in remission after non-bacterial AECOPD (p<0,05).

The absolute number of induced sputum macrophages in both AECOPD groups, due to relative predominance of induced sputum neutrophils, was lower than in both healthy groups (p<0.05). The amount of induced sputum macrophages was higher during bacterial AECOPD than during non-bacterial AECOPD and remission of COPD after bacterial AECOPD (p<0.05).

The amount of peripheral blood neutrophils and monocytes in relative and absolute values was higher in patients with bacterial AECOPD than those with non-bacterial AECOPD, remission after bacterial AECOPD and both groups of healthy subjects (p<0.05) (Table 3.2.1 and 3.2.2).

Table 3.2.1. Induced sputum cell composition in patients with COPD

exacerbation and in remission and healthy subjects of comparison

Variable Bacterial AECOPD (n=23) Non- bacterial AECOPD (n=27) Bacterial rCOPD (n=23) Non- bacterial rCOPD (n=27) „Healthy“ smokers (n=10) Healthy Non- smokers (n=10) Induced sputum neutrophils, ×106 /mL 1.94* (1.67–2.40) 1.68 (1.48–2.00) 1.76** (1.62–2.30) 1.66 (1.46–1.95) 0.55# (0.47–0.78) 0.45# (0.34–0.50) Induced sputum macrophages ×106/mL 0.75* (0.38–0.99) 0.58 (0.32–0.84) 0.73** (0.52–0.92) 0.56 (0.21–0.75) 0.81‡ (0.65–0.96) 0.79‡ (0.76–1.14) Data are shown as median (min.-max.). AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD.

*p<0.05, compare with bacterial rCOPD and non-bacterial AECOPD; **p<0.05, compare

with non-bacterial rCOPD; #p<0.05, compared with all COPD patients groups; ‡p<0.05,

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Table 32.2. Peripheral blood cell composition in patients with COPD exacerbation and in remission and healthy subjects of comparison Bacterial AECOPD (n=23) Non-bacterial AECOPD (n=27) Bacterial rCOPD (n=23) Non-bacterial rCOPD (n=27) „Healthy“ smokers (n=10) Healthy Non-smokers (n=10) Peripheral blood neutrophils, ×109/L, % 4.87* (4.67–5.44) 77.23* (74.58–79.57) 4.37 (4.21– 4.58) 62.04 (58.23– 63.57) 4,86** (4.62–5.30) 75.64** (67.87–77.58) 4.22 (4.15– 4.95) 61.24 (57.47– 62.34) 3.75# (3.55–3.81) 56.81# (53.24–58.04) 3.65† (3.40–3.78) 56.75† (52.47–57.84) Peripheral blood monocytes ×109/L, % 0.71* ‡ (0.67–0.79) 7.34* ‡ (6.97–7.54) 0.57 (0.48–0.67) 7.01 (6.57–7.27) 0.65** (0.62–0.70) 7.27** (6.84–7.40) 0.56 (0.47–0.62) 7.01 (6.58–7.25) 0.47# (0.39–0.48) 6.37# (6.29–6.74) 0.45# (0.41–0.49) 6.36# (6.27–6.68) Data are shown as median (min.-max.). AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD.

*p<0.05, compared with bacterial rCOPD; **p<0.05, compared with non-bacterial rCOPD;

#

p<0.05, compared with all COPD patients groups and healthy non-smokers; †p< 0.05, compared with all COPD patients groups and „healthy“ smokers; ‡p<0.05, compared with non-bacterial AECOPD.

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3.3. Relationships between induced sputum neutrophils, macrophages and peripheral blood neutrophils and pulmonary function test and

smoking history

The relationship between induced sputum neutrophils and macrophages and peripheral blood neutrophils and pulmonary function test and smoking history is represented in Table 3.3.1.

Table 3.3.1. Relations between induced sputum neutrophils and macro-

phages and peripheral blood neutrophils and FEV1 and smoking intensity in all investigational groups

Count of cells Subjects FEV 1 (L) p Smoking hystory (packyears) p Induced sputum neutron- phils (×106/ml) Bacterial AECOPD (n=23) r=–0,571 p=0,041 r=–0,584 p=0,005 Non-bacterial AECOPD (n=27) r=–0,644 p=0,024 r=–0,491 p=0,026 Bacterial rCOPD (n=23) r=–0,485 p=0,025 r=–0,517 p=0,043 Non-bacterial rCOPD (n=27) r=–0,593 p=0,034 r=–0,482 p=0,030 “Healthy” smokers (n=10) r=–0,344 p=0,044 r=–0,307 NS Healthy Non-smokers (n=10) r=–0,331 NS Macro- phages (×106/ml) Bacterial AECOPD (n=23) r=–0,684 p=0,011 r=0,514 p=0,039 Non-bacterial AECOPD (n=27) r=–0,777 p=0,019 r=–0,611 p=0,017 Bacterial rCOPD (n=23) r=–0,554 p=0,021 r=–0,541 p=0,024 Non-bacterial rCOPD (n=27) r=–0,517 p=0,041 r=0,471 p=0,006 “Healthy” smokers (n=10) r=–0,422 p=0,021 r=–0,250 NS Healthy Non-smokers (n=10) r=–0,243 NS Periphera l blood neutrophils 9 (×10 /L) Bacterial AECOPD (n=23) r=–0,450; p=0,004 r=–0,247; p=0,043 Non-bacterial AECOPD (n=27) r=–0,574; p=0,003 r=–0,544; p=0,027 Bacterial rCOPD (n=23) r=–0,450; p=0,002 r=–0,272; p=0,035 Non-bacterial rCOPD (n=27) r=–0,457; p=0,001 r=–0,311; p=0,043 “Healthy” smokers (n=10) r=–0,288; p=0,031 r=–0,241; NS Healthy Non-smokers (n=10) r=–0,208; NS

AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD; r – Spearman correlation.

3.4. Induced sputum cellular activity

Apoptosis of induced sputum neutrophils and macrophages

Apoptosis of induced sputum neutrophils and macrophages signify- cantly reduced during bacterial AECOPD compare with non-bacterial AECOPD and remission after bacterial AECOPD and both healthy patients groups (p<0.05). Reduced apoptosis obtained during non-bacterial AECOPD compare with remission after non-bacterial AECOPD and both healthy groups (p<0.05) (Fig. 3.4.1).

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Fig. 3.4.1. Apoptosis of induced sputum neutrophils (A) and

macrophages (B)

Data are shown as median (min.-max.). AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD. #p<0.05, compared with all AECOPD and rCOPD

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Phagocytosis of induced sputum neutrophils

S. aureus bacteria stimulated neutrophil phagocytosis in dose de-

pendent way in all study groups but significant difference we found after stimulation with 167 S. aureus bacteria per neutrophil and the same con- centration per macrophage. The phagocytosis in induced sputum neutrophils and macrophages during bacterial AECOPD was weaker than in non- bacterial AECOPD and remission after bacterial AECOPD (p<0.05). We obtained that phagocytosis significantly intensifying in remission of COPD after non-bacterial AECOPD comparing with non-bacterial AECOPD (p<0.05). All COPD patients compare with both healthy subjects groups shown weakest phagocytosis of induced sputum neutrophils and macrophages (p<0.05) (Fig. 3.4.2).

The production of reactive oxygen species in induced sputum neutrophils and macrophages

Spontaneous production of ROS in induced sputum neutrophils and macrophages was higher during bacterial AECOPD compared with non- bacterial AECOPD (p<0.05) and remission after bacterial AECOPD (p<0.0001). A higher ROS production was obtained in non-bacterial AECOPD patients group compared with remission after non-bacterial AECOPD, (p<0.0001). We found difference between smokers and non- smokers healthy subjects (p<0.0001) (Fig. 3.4.3).

PMA induced ROS production in induced sputum neutrophils and macrophages in dose dependent way in all studied patients groups. We had significant difference between study groups using higher PMA concentration (3, 10 and 30 nM of PMA). The higher ROS production of induced sputum neutrophils and macrophages was found during bacterial AECOPD than non-bacterial AECOPD (p<0.05). The higher ROS production was seen in non-bacterial AECOPD than remission after non-bacterial AECOPD (p<0.05). According smoking status, a higher ROS production was obtained in “healthy” smokers compared with healthy non-smokers (p<0.05).

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#

p<0.05, compared with all AECOPD and rCOPD groups.

Fig. 3.4.2. Phagocytosis of induced sputum neutrophils (A) and

macrophages (B) after activated with 167 S. aureus bacteria per neutrophil or macrophage

Data are shown as median. AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD. #p<0.05, compared with all AECOPD and rCOPD groups.

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#

p<0.05, compared with all AECOPD and rCOPD groups.

Fig. 3.4.3. Spontaneous production of reactive oxygen species

in induced sputum neutrophils (A) and macrophages (B)

Data are shown as median (min.-max.).

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S. aureus bacteria, as well as PMA, stimulated ROS production in

dose dependent way in all study groups but significant difference was ob- tained using higher dose of S. aureus bacteria per neutrophil or macro- phage. A higher ROS production, compared with non-bacterial AECOPD, was obtained in induced sputum neutrophils during bacterial AECOPD (p<0.05). The ROS production significantly decreased in remission of COPD after bacterial AECOPD compare with bacterial AECOPD (p<0.05). Induced sputum neutrophils and macrophages produced higher ROS in “healthy” smokers group than healthy non-smokers at 18, 56 and 167 S. aureus bacteria per neutrophil or macrophage concentrations (p<0.05).

S. aureus bacteria induced weaker ROS production in induced sputum

neutrophils and macrophages than PMA (p<0.05).

3.5. Peripheral blood cellular activity Apoptosis of peripheral blood neutrophils and monocytes

The most reduced apoptosis was obtained during bacterial and non- bacterial AECOPD compare with remission of COPD after bacterial and non-bacterial AECOPD (p<0.0001). We obtained significantly reduced apoptosis in bacterial AECOPD than non-bacterial AECOPD in peripheral blood neutrophils and monocytes (p=0.0028 and p<0.0001 respectively). No differences according to smoking status between healthy groups were obtained (Fig. 3.5.1).

Chemotaxis of peripheral blood neutrophils and monocytes

IL-8 stimulated a higher chemotaxis of peripheral blood neutrophils during bacterial AECOPD compare with non-bacterial AECOPD (p=0.013). A higher chemotaxis was obtained during both AECOPD compare with remission after AECOPD (in bacterial and nonbacterial groups p<0.0001). According smoking status, higher peripheral blood neutrophil chemotaxis was in “healthy” smokers group than healthy non-smokers only stimulating with the highest concentration of IL-8 (100 ng/mL) (p=0.0002) (Fig. 3.5.2 A).

Significant difference between all study groups was found stimulating peripheral blood monocytes 100 ng/mL concentration of C5a. The higher chemotaxis was assessed during bacterial AECOPD compare with non-

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Fig. 3.5.1. Apoptosis of peripheral blood neutrophils (A)

and monocytes (B)

Data are shown as median (min.-max.). AECOPD – acute exacerbation of COPD; rCOPD (bact.) – remission of COPD after bacterial AECOPD; rCOPD (non-bact.) –

remission of COPD after non-bacterial AECOPD. #p<0.05, compared with all AECOPD and rCOPD groups.

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bacterial AECOPD (p<0.0001) and remission after bacterial AECOPD (p<0.0001) as well as during non-bacterial AECOPD compare with remission after non-bacterial AECOPD (p<0.0001). Higher peripheral monocytes chemotaxis was obtain in “healthy” smokers than non-smokers (p=0.0063) but chemotaxis was lower in both healthy groups compare with all COPD groups (p<0.05) (Fig. 3.5.2 B).

Phagocytosis of peripheral blood neutrophils

Higher phagocytosis was obtained after stimulation peripheral blood neutrophils with highest 167 S. aureus bacteria per neutrophil concentra- tion. The weaker phagocytosis was obtained in bacterial AECOPD group than non-bacterial AECOPD (p<0.0001). Phagocytosis increased in remission of COPD compare with AECOPD (p<0.0001). The most intensive phagocytosis was obtained in healthy subjects groups compare with all COPD groups (p<0.05). No difference according smoking status in peripheral blood neutrophils were obtained (Fig. 3.5.3).

The production of reactive oxygen species in peripheral blood neutrophils

A higher spontaneous ROS production was obtained during AECOPD compare with remission after AECOPD (p<0.0001) but during bacterial AECOPD ROS production increased much more than during non-bacterial AECOPD (p<0.0001). Smoking increased ROS production in peripheral blood neutrophils compare with non-smokers (p<0.0001). Lowest ROS production was obtain in both healthy groups compare with all COPD patients groups (p<0.05) (Fig. 3.5.4).

Peripheral blood neutrophils produced highest ROS using 30 nM concentration of PMA in all study groups. PMA induced higher ROS production in bacterial AECOPD than in non-bacterial (p<0.05) and remission after bacterial AECOPD groups (p<0.05). We assessed that increased ROS production was in all COPD patients groups compare with healthy smokers and non-smokers (p<0.05). Peripheral blood neutrophils produce higher ROS in smokers than non-smokers (p<0.05).

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Fig. 3.5.2. Chemotaxis of peripheral blood neutrophils stimulated

with 100 mg/mL concentration of IL-8 (A) and monocytes stimulated with 100 mg/mL concentration of C5a (B)

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Fig. 3.5.3. Phagocytosis of peripheral blood neutrophils activated with

167 S. aureus bacteria per neutrophil

Fig. 3.5.4. Spontaneous production of reactive oxygen species in

peripheral blood neutrophils

Data are shown as median (min.-max.). AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD. #p<0.05, compared with all AECOPD and rCOPD

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S. aureus bacteria stimulated ROS production in peripheral blood

neutrophils as biological factor. S. aureus induced ROS production in dose dependent way in all study groups. Differences between all groups were observed using high concentration of S. aureus bacteria per neutrophil (56 and 167 S. aureus bacteria per neutrophil concentration). The highest ROS production in peripheral blood neutrophils was obtained during bacterial AECOPD compare with non-bacterial AECOPD (p<0.05). The ROS production reduced in remission of COPD after AECOPD (p<0.05). Peri- pheral blood neutrophils produced lower ROS in healthy subjects group compare with all COPD groups (p<0.05) but higher in smokers than non- smokers (p<0.05).

3.6. Concentration of interleukin-8 in induced sputum and serum and C reactive protein in peripheral blood and relationship with peripheral blood neutrophil chemotaxis and pulmonary function

test and smoking hystory

The highest concentration of IL-8 was in induced sputum than in peripheral blood in all study groups. Highest IL-8 concentration was obtain in AECOPD groups compare with remission after AECOPD and healthy groups (p<0.05). The highest IL-8 concentration was assessed during bacterial AECOPD than non-bacterial AECOPD (p<0.05) (Table 3.6.1).

C reactive protein concentration in peripheral blood was obtain during bacterial AECOPD than non-bacterial AECOPD (p<0.05) and remission after bacterial AECOPD (p<0.05). No differences were detected compare- ing CRP concentrations in “healthy” smokers and non-smokers groups (Table 3.6.1).

The relationships between CRP levels and pulmonary function test and smoking history are presented in Table 3.6.2.

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Subjects FEV1 (L) p Smoking hystory (packyears) p CRB (mg/l) Bacterial AECOPD (n=23) r=0,341 p=0,378 r=0,247 NS Non-bacterial AECOPD (n=27) r=0,421 p=0,478 r=0,278 NS Bacterial rCOPD (n=23) r=0,257 p=0,674 r=0,362 NS Non-bacterial rCOPD (n=27) r=0,354 p=0,247 r=0,243 NS „Healthy“ smokers (n=10) r=0,254 p=0,087 r=0,326 NS Healthy Non-smokers (n=10) r=0,547 p=0,247 – –

Table 3.6.1. Concentration of interleukin-8 in induced sputum and serum

and C reactive protein in peripheral blood

Variable Bacterial AECOPD (n=23) Non- bacterial AECOPD (n=27) Bacterial rCOPD (n=23) Non- bacterial rCOPD (n=27) „Healthy“ smokers (n=10) Healthy Non- smokers (n=10) IL-8, pg/ml in peripheral blood 128,8 ±12,4* 58,2±7,4 89,8±8,1** 47,8±4,8 13,7±5,6# 7,5±2,4† IL-8, pg/ml in induced sputum 475,7±15,4*‡ 357,7±31,2‡ 468,7±15,1**‡ 297,8±22,7‡ 67,8±7,8†‡ 52,7±6,8†‡ CRP, mg/L 24,9±16,7* 6,7±4,7 16,4±5,4** 5,4±2,7 3,7±2,8†† 1,2±0,7††

Data are present as median±SD. SD – standard deviation; AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD; IL – interleukin; CRP – C reactive protein.

*p<0.05, compared with non-bacterial AECOPD and bacterial rCOPD, **p<0.05, compa-

red with non-bacterial rCOPD, #p<0.05, compared with all COPD groups and healthy non-

smokers, †p<0.05, compared with all COPD groups; ‡p<0,05, compare with serum IL-8;

‡‡

p<0,05, compared with both AECOPD groups.

Table 3.6.1. Relations between CRP levels and FEV1 and smoking history

AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD; FEV1 – forced expiratory volume in 1 sec.

We found significant correlations between amount of induced sputum neutrophils and migrated peripheral blood neutrofils in all COPD patients groups except both healthy subjects groups (Table 3.6.3).

Interleukin-8 concentration in induced sputum and serum significant correlated with migrated peripheral blood neutrophils in all COPD patients groups. In healthy patients groups the correlation between IL-8 concentration in induced sputum and serum was not significant (Table 3.6.3).

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Table 3.6.3. Relations between migrated peripheral blood neutrophil and

interleukin-8 levels in induced sputum and serum Subjects Migrated peripheral blood neutrophils (×106/mL) p Induced sputum neutrophils (×106/ml) Bacterial AECOPD (n=23) r=0,681 p=0,004 Non-bacterial AECOPD (n=27) r=0,754 p=0,003 Bacterial rCOPD (n=23) r=0,572 p=0,025 Non-bacterial rCOPD (n=27) r=0,542 p=0,015 „Healthy“ smokers (n=10) r=0,321 p=0,254 Healthy Non-smokers (n=10) r=0,247 p=0,125 IL-8 concentration in induced sputum (pg/mL) Bacterial AECOPD (n=23) r=0,357 p=0,027 Non-bacterial AECOPD (n=27) r=0,337 p=0,035 Bacterial rCOPD (n=23) r=0,421 p=0,018 Non-bacterial rCOPD (n=27) r=0,304 p=0,039 „Healthy“ smokers (n=10) r=0,257 p=0,042 Healthy Non-smokers (n=10) r=0,308 p=0,113 IL-8 concentration in serum (pg/mL) Bacterial AECOPD (n=23) r=0,654 p=0,001 Non-bacterial AECOPD (n=27) r=0,627 p=0,001 Bacterial rCOPD (n=23) r=0,542 p=0,004 Non-bacterial rCOPD (n=27) r=0,534 p=0,015 „Healthy“ smokers (n=10) r=0,432 p=0,159 Healthy Non-smokers (n=10) r=0,352 p=0,194 AECOPD – acute exacerbation of COPD; rCOPD – remission of COPD;

IL – interleukin.

CONCLUSIONS

1. Increased count of induced sputum neutrophils and macrophages as well peripheral blood neutrophils and monocytes during bacterial and non-bacterial acute exacerbation of chronic obstructive pulmonary disease related with impaired pulmonary function and smoking history. 2. Induced sputum neutrophils and macrophage apoptosis and phago-

cytosis were weaker, but production of reactive oxygen species was strongly activated during bacterial acute exacerbation of chronic

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obstructive pulmonary disease than non-bacterial acute exacerbation of chronic obstructive pulmonary disease.

3. Peripheral blood neutrophil and monocyte activity was impaired during bacterial acute exacerbation of chronic obstructive pulmonary disease: neutrophil and monocyte apoptosis and phagocytosis was decreased, while the production of reactive oxygen species was significantly increased compare with non-bacterial acute exacerbation of chronic obstructive pulmonary disease.

4. Higher concentration of IL-8 in peripheral blood and induced sputum during bacterial and non-bacterial acute exacerbation of chronic obstructive pulmonary disease was related with chemotaxis of peripheral blood neutrophils. Higher concentration of C reactive protein was not related with pulmonary function and smoking history.

PRACTICAL SIGNIFICANCE OF THE STUDY

The results of the study demonstrated more information about the non- specific inflammation during acute exacerbation of chronic obstructive pulmonary disease.

Analysis of inflammatory cells activity during bacterial and non- bacterial acute exacerbation of chronic obstructive pulmonary disease gave opportunity investigate direct effect of infection agent on cell apoptosis, chemotaxis, phagocytosis and ROS production activated by biological and chemical factors.

It is clear that is important to find out the agent of acute exacerbation of chronic obstructive pulmonary disease. Bacterial agent causes much intensive inflammation in the lungs. Increased inflammation damaged on the lung tissue what cause further development of emphysema and loss of pulmonary function and worse health status of COPD patients.

LIST OF AUTHOR’S PUBLICATIONS

1. Lavinskienė, Simona; Vaitkus, Mindaugas; Biekšienė, Kristina; Je- roch, Jolanta; Malakauskas, Kęstutis; Sakalauskas, Raimundas. Kraujo neutrofilų fagocitinis aktyvumas in vitro sergant lėtine obstrukcine plaučių liga = Neutrophil phagocytic activity during chronic obs-

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tructive pulmonary disease / Simona Lavinskienė, Mindaugas Vaitkus, Kristina Biekšienė, Jolanta Jeroch, Kęstutis Malakauskas, Raimundas Sakalauskas // Lietuvos bendrosios praktikos gydytojas. Kaunas : Vitae Litera. (Moksliniai darbai). ISSN 1392-3218. 2011, t. 15, Nr. 9, p. 651-655.

2. Vaitkus, M., Lavinskiene, S., Bieksiene, K., Barkauskiene, D., Jeroch, J., Sakalauskas, R. Reactive oxygen species in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbation of chronic obstructive pulmonary disease. Inflammation, 2013. 36(6): p. 1485-93.

3. Lavinskienė, Simona; Vaitkus, Mindaugas; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Neutrophil phagocytic activity in AECOPD / Simona Lavinskiene, Mindaugas Vaitkus, Kristina Bieksiene, Jolanta Jeroch, Raimundas Sakalauskas // European Respiratory Journal : 21st Annual Congress Amsterdam, The Nether- lands 24–28 September 2011: Abstracts / European Respiratory Society; Published for the European Respiratory Society by Maney Publishing. (89. COPD: human studies.). ISSN 0904-1850. 2011, vol. 38, suppl. 55, p. 121s, no. P738.

4. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Lėtinės obstrukcinės plaučių ligos paūmėjimo įtaka reaktyviųjų deguonies formų (ROS) susidary- mui neutrofiluose / Mindaugas Vaitkus, Simona Lavinskienė, Kristina Biekšienė, Jolanta Jeroch, Raimundas Sakalauskas // Pulmonologija, imunologija ir alergologija : Lietuvos sveikatos mokslų universiteto ir Lietuvos pulmonologų ie alergologų draugijos konferencijos „Pulmo- nologija, alergologija ir klinikinė imunologija 2011“ : mokslinės tezės : 2011 m. balandžio 15 d. Kaunas : Medicinos spaudos namai. (Moks- linės tezės.). ISSN 1822-6884. 2011, Nr. 2(9), p. 32-33.

5. Lavinskienė, Simona; Vaitkus, Mindaugas; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Kraujo neutrofilų fagocitinis aktyvumas lėtinės obstrukcinės plaučių ligos paūmėjimo metu / Simona Lavinskienė, Mindaugas Vaitkus, Kristina Biekšienė, Jolanta Jeroch, Raimundas Sakalauskas // Pulmonologija, imunologija ir alergologija : Lietuvos sveikatos mokslų universiteto ir Lietuvos pulmo- nologų ir alergologų draugijos konferencijos „Pulmonologija, alergologija ir klinikinė imunologija 2011“ : mokslinės tezės : 2011 m. ba- landžio 15 d. Kaunas : Medicinos spaudos namai. (Mokslinės tezės.). ISSN 1822-6884. 2011, Nr. 2(9), p. 29 : pav.

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6. Lavinskienė, Simona; Vaitkus, Mindaugas; Biekšienė, Kristina; Je- roch, Jolanta; Malakauskas, Kęstutis; Sakalauskas, Raimundas. Kraujo neutrofilų fagocitinis aktyvumas in vitro sergant lėtine obstrukcine plaučių liga = Neutrophil phagocytic activity during chronic obstructive pulmonary disease / Simona Lavinskienė, Mindaugas Vaitkus, Kristina Biekšienė, Jolanta Jeroch, Kęstutis Malakauskas, Raimundas Sakalauskas // Lietuvos bendrosios praktikos gydytojas. Kaunas : Vitae Litera. (Moksliniai darbai). ISSN 1392-3218. 2011, t. 15, Nr. 9, p. 651-655.

7. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Influence of AECOPD to ROS production in neutrophils / Mindaugas Vaitkus, Simona Lavins- kiene, Kristina Bieksiene, Jolanta Jeroch, Raimundas Sakalauskas // European Respiratory Journal : 21st Annual Congress Amsterdam, The Netherlands 24–28 September 2011: Abstracts / European Res- piratory Society; Published for the European Respiratory Society by Maney Publishing. (89. COPD: human studies.). ISSN 0904-1850. 2011, vol. 38, suppl. 55, p. 120s, no. P736.

8. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Reaktyvių deguonies formų susidarymas periferinio kraujo ir skreplių neutrofiluose paūmėjus LOPL / Mindaugas Vaitkus, Simona Lavinskienė, Kristina Biekšienė, Jolanta Jeroch, Raimundas Sakalauskas // Pulmonologija, imunologija ir alergologija : PIA : Lietuvos sveikatos mokslų universiteto ir Lie- tuvos pulmonologų ir alergologų draugijos konferencijos „Pulmono- logija, alergologija ir klinikinė imunologija 2012“ mokslinės tezės : 2012 m. gegužės 4 d. Kaunas : Medicinos spaudos namai. (Mokslinės tezės.). ISSN 1822-6884. 2012, Nr. 1(10), p. 67.

9. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Reaktyvių deguonies formų susidarymas neutrofiluose paūmėjus LOPL : Mindaugas Vaitkus, Si- mona Lavinskienė, Kristina Biekšienė, Jolanta Jeroch, Raimundas Sa- kalauskas // V nacionalinė doktorantų mokslinė konferencija „Moks- las – sveikatai“ [elektroninis išteklius] : 2012 m. balandžio 11 d. : Konferencijos tezių rinkinys / Lietuvos sveikatos mokslų universitetas. Kaunas : Lietuvos sveikatos mokslų universitetas, 2011. (Sekcija „Pulmonologija ir alergologija“.). ISBN 978-9955-15-232-3 (CD) ; 978-9955-15-231-6 (internete). 1 skelb, p. 34/75, Nr. 2.

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10. Lavinskienė, Simona; Vaitkus, Mindaugas; Jeroch, Jolanta; Biekšienė, Kristina; Sakalauskas, Raimundas. Reactive oxygen species in neutrophils during acute exacerbation of COPD : [elektroninis iš- teklius] / S. Lavinskiene, M. Vaitkus, J. Jeroch, K. Bieksiene and R. Sakalauskas // CYTO 2012 : ISAC XXVII International congress : program and abstracts : Leipzig, Germany 23-27 June, 2012 : [elektroninis išteklius]. p. 222, poster B 365/B259.

11. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Analysis of reactive oxygen species in sputum neutrophils during acute exacerbation of COPD : [elektroninis išteklius] / M. Vaitkus, S. Lavinskiene, K. Bieksiene, J. Jeroch, R. Sakalauskas // ERS 2012 : 22nd European Respiratory Society Annual Congress : programme : Vienna, Austria September 1- 5, 2012 : [electronic resource]. poster P793.

12. Vaitkus, Mindaugas; Lavinskienė, Simona; Barkauskienė, Diana; Biekšienė, Kristina; Jeroch, Jolanta; Sakalauskas, Raimundas. Reac- tive oxygen species in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbation of chronic obstructive pulmonary disease / Vaitkus M, Lavinskiene S, Barkauskiene D, Biek- siene K, Jeroch J, Sakalauskas R // Inflammation. New York, NY : Kluwer Academic/Plenum Publishers. ISSN 0360-3997. 2013, vol. 36, no. 6, p. 1485-1493 : pav, lent.

13. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Monocitų apoptozė ir chemotaksis bei makrofagų fagocitozė LOPL paūmėjimo metu / Min- daugas Vaitkus, Simona Lavinskienė, Kristina Biekšienė, Jolanta Je- roch, Raimundas Sakalauskas // Pulmonologija, imunologija ir alergologija : PIA : Lietuvos sveikatos mokslų universiteto ir Lietuvos pulmonologų ir alergologų draugijos konferencijos „Pulmonologija, alergologija ir klinikinė imunologija 2013“ mokslinės tezės : 2013 m. gegužės 3 d. Kaunas : Medicinos spaudos namai. (Mokslinės tezės.). ISSN 1822-6884. 2013, Nr. 1(12), p. 39-40.

14. Vaitkus, Mindaugas; Lavinskienė, Simona; Biekšienė, Kristina; Je- roch, Jolanta; Sakalauskas, Raimundas. Influence of AECOPD to monocytes apoptosis and chemotaxis and macrophages phagocytosis / Mindaugas Vaitkus, Simona Lavinskiene, Kristina Bieksiene, Jolanta Jeroch, Raimundas Sakalauskas // 11th ERS lung science conferece „Early origins and mechanisms of chronic lung disease“ : March 15- 17, 2013, Estoril, Portugal. p. 151, PP141.

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SUMMARY IN LITHUANIAN

Darbo tikslas

Įvertinti nespecifinį uždegimą paūmėjus lėtinei obstrukcinei plaučių ligai.

Darbo uždaviniai

1. Nustatyti periferinio kraujo ir indukuotų skreplių ląstelių sudėties ypatumus ir įvertinti galimas sąsajas su plaučių funkcijos rodikliais bakterinio ir nebakterinio lėtinės obstrukcinės plaučių ligos paūmėjimo metu.

2. Ištirti sergančiųjų bakterinės kilmės lėtinės obstrukcinės plaučių ligos paūmėjimo indukuotų skreplių neutrofilų ir makrofagų apoptozę, fa- gocitozę ir reaktyvių deguonies formų susidarymo ypatumus bei paly- ginti su nebakteriniu paūmėjimu.

3. Įvertinti sergančiųjų bakterinės ir nebakterinės kilmės lėtinės obs- trukcinės plaučių ligos paūmėjimo periferinio kraujo neutrofilų ir mo- nocitų apoptozę ir chemotaksį, bei periferinio kraujo neutrofilų fago- citozės ir reaktyvių deguonies formų susidarymo ypatumus.

4. Ištirti sergančiųjų bakterinės ir nebakterinės kilmės lėtinės obstrukcinės plaučių ligos paūmėjimu interleukino-8 koncentraciją ir galimas sąsajas su periferinio kraujo neutrofilų chemotaksiu bei C reaktyviojo baltymo koncentracijos periferinio kraujo serume sąsajas su plaučių funkcija ir rūkymo intensyvumu.

Metodika:

Tyrimas atliktas gavus Kauno regioninio biomedicininių tyrimų etikos komiteto (protokolo Nr. P1-48/2004, versija 4, 2010) leidimą.

Įtraukti 50 pacientų sergančių lėtinės obstrukcinės plaučių ligos (LOPL) paūmėjimu ir tie patys pacientai ligos remisijoje bei 10 rūkančių sveikų ir 10 nerūkančių sveikų asmenų, kaip kontrolinė grupė. LOPL diag- nozuota remiantis Pasaulinės lėtinės obstrukcinės plaučių ligos iniciatyvos (GOLD) kriterijais.

Pacientams, kuriems diagnozuotas LOPL paūmėjimas buvo skiriamas gydymas inhaliaciniais vaistais bei sisteminio poveikio gliukokortikoste- roidais pagal Pasaulinės lėtinės obstrukcinės plaučių ligos iniciatyvos (GOLD) rekomendacijas. Nustačius infekcinį sukėlėją papildomai skirti antibiotikai (β laktaminiai antibiotikai). Išnykus LOPL paūmėjimo požy-

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miams tęstas gydymas inhaliuojamais vaistais atitinkamai pagal ligos sun- kumą remiantis. Tie patys pacientai atvyko antram vizitui po 35 dienų, esant ligos remisijai.

Uždegimo ląstelių tyrimui buvo paimtas kraujas iš periferinės venos bei indukuoti skrepliai. Indukuoti skrepliai tirti mikrobiologiniu tyrimu – pacientai, kuriems buvo išskirtas sukėlėjas, buvo suskirstyti į bakterinio LOPL paūmėjimo pacientų grupę, neišskyrus sukėlėjo – nebakterinio LOPL paūmėjimo grupė. Tikslu ištirti skreplių neutrofilus ir makrofagus visiems tiriamiesiems buvo indukuoti skrepliai hipertoninių natrio chlorido tirpalu. Vertintas indukuotų skreplių neutrofilų ir makrofagų bei periferinio kraujo neutrofilų ir monocitų absoliutus ir santykinis skaičius. Interleukino-8 (IL- 8) koncentracija vertinta kraujo serume ir indukuotuose skrepliuose naudojant ELISA metodą, C reaktyvinio baltymo (CRB) koncentracija vertinta turbodimetriniu metodu.

Indukuotų skreplių neutrofilai išskirti naudojant fosfatinį buferį, o makrofagai – magnetinės separacijos rinkinį. Periferinio kraujo neutrofilai išskirti naudojant aukšto tankio gradientą ir lizuojant eritrocitus, o indu- kuotų skreplių monocitai išskirti naudojant magnetinės separacijos rinkinį.

Uždegimo ląstelių funkcijos kaip apoptozė, chemotaksis, fagocitozė ir reaktyvių deguonies formų (ROS) susidarymas analizuoti naudojant tėkmės citometrą (FACSCalibur, BD Biosciences, JAV).

Apoptozė vertinta naudojant aneksiną ir propidžio jodidą. Ląstelės inkubuojamos 15 minučių ir vertinama tėkmės citometrijos metodu. Che- motaksis vertintas naudojant chemotaksio kamera, kurios apatiniai šulinėliai užpildomi chemotaktinėmis medžiagomis (IL-8 – periferinio kraujo neutro- filų, C5a – monocitų vertinimui), o viršutiniai – periferinio kraujo neutrofilų ir monocitų ląstelėmis. Viršutiniai šulinėliai nuo apatinių atskirti membrana. Per membraną migravusių ląstelių skaičiui nustatyti naudotas rinkinys, kurį sudaro mikro-rutuliukai, bei propidžio jodidas su tiazolo oranžo dažu. Ląstelių populiacijų ypatumai analizuoti tėkmės citometru. Fagocitozė vertinta ląsteles aktyvuojant skirtingomis S. aureus bakterijų koncentracijomis. Santykinis fagocitinio aktyvumo intensyvumas vertintas tėkmės citometrijos metodu. Santykinis ROS susidarymas ištirtas naudojant skirtingas biologinio faktoriaus S. aureus ir cheminio faktoriaus PMA koncentracijas aktyvintose ląstelėse tėkmės citometrijos metodu.

Statistinė analizė atlikta naudojant GraphPad Prism 6 for Windows programos paketą. Planuojant mokslinį darbą, reikalingas atvejų skaičius buvo apskaičiuotas naudojant GraphPad StatMate 2 for Windows progra-

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mą. Statistiniu metodu pasirinktas galingumas >0,8. Reikšmingumo lygmuo tikrinant statistines hipotezes pasirinktas 0,05.

Rezultatai:

Vertinant visų tiriamųjų grupių amžių, kūno masės indeksą ir ku- muliacinį rūkymo intensyvumą reikšmingų skirtumų rasta nebuvo. Nusta- tyta, jog bakterinio LOPL paūmėjimo metu reikšmingai sumažėja plaučių funkcijos rodikliai FEV1 ir FVC lyginant su nebakteriniu paūmėjimu

(p<0,05) ir ligos remisija (p<0,05). Analizuojant Šv. Jurgio ligoninės kvė- pavimo klausimyną nustatytas reikšmingai didesnis balų skaičius bakterinio LOPL paūmėjimo metu nei nebakterinio (p<0,05) ir ligos remisijoje (p<0,05).

Vyraujančios ląstelės indukuotuose skrepliuose – neutrofilai. Nusta- tytas reikšmingas šių ląstelių skaičiaus padidėjimas bakterinio LOPL paū- mėjimo metu nei nebakterinio (p<0,05) ir remisijoje (p<0,05). Panašūs duomenys gauti nagrinėjant indukuotų skreplių makrofagų skaičių – daugiausia nustatyta bakterinio LOPL paūmėjimo metu. Periferinio kraujo neutrofilų ir monocitų skaičius reikšmingai padidėja bakterinio LOPL paūmėjimo metu lyginant su nebakteriniu (p<0,05) ir ligos remisija (p<0,05).

Nustatytos atvirkštinės sąsajos tarp indukuotų skreplių neutrofilų ir makrofagų bei periferinio kraujo neutrofilų ląstelių skaičiaus ir plaučių funkcijos rodiklio FEV1 bakterinio ir nebakterinio LOPL paūmėjimų bei

remisijų grupėse. Analizuojant indukuotų skreplių neutrofilų ir makrofagų bei periferinio kraujo neutrofilų ląstelių skaičių ir kumuliacinį rūkymo in- tensyvumą gautos tiesioginės sąsajos. Sveikų asmenų grupėse sąsajų negauta.

Indukuotų skreplių neutrofilų ir makrofagų apoptozė ir fagocitozė reikšmingai susilpnėjo bakterinio LOPL paūmėjimo metu lyginant su nebakteriniu paūmėjimu, ligos remisija bei visomis sveikų asmenų grupėmis (p<0,05). Analizuojant savaiminį ir aktyvuotą S. aureus bei PMA reaktyvių deguonies formų susidarymą nustatyta, jog daugiausia ROS susidaro LOPL paūmėjimo metu ir ypač bakterinio LOPL paūmėjimo lyginant su nebakteriniu (p<0,05) ir remisija (p<0,05).

Nustatyta, jog periferinio kraujo neutrofilų ir monocitų apoptozė susilp- nėja bakterinio LOPL paūmėjimo metu lyginant su nebakteriniu (p<0,05) ir ligos remisija (p<0,05). Chemotaksis reikšmingai sustiprėja labiau bakterinio nei nebakterinio LOPL paūmėjimo metu (p<0,05). LOPL remisijų metu stebėtas chemotaksio susilpnėjimas lyginant su LOPL paūmėjimu (p<0,05), bet išlieka stipresnis lyginant su sveikais asmenimis (p<0,05). Periferinio

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kraujo neutrofilų fagocitozė sumažėja reikšmingai labiau bakterinio LOPL paūmėjimo metu nei nebakterinio (p<0,05). Vertinant ligos remisijų grupės tiriamuosius fagocitozė sustiprėja, bet išlieka silpnesnė nei sveikų asmenų (p<0,05). ROS susidarymas periferinio kraujo neutrofiluose intensyviausias buvo bakterinio LOPL paūmėjimo metu lyginant su nebakteriniu, panašūs duomenys gauti aktyvuojant ląsteles S. aureus bakterijomis ir PMA.

Didžiausia IL-8 koncentracija nustatyta indukuotuose skrepliuose ir ypač bakterinio LOPL paūmėjimo metu lyginant su nebakteriniu. Reikšmin- gas skirtumas nustatytas lyginant IL-8 koncentraciją indukuotuose skrepliuose ir periferiniame kraujyje – daugiau IL-8 pagaminama indukuotuose skrepliuose (p<0,05). Didžiausia CRB koncentracija nustatyta LOPL paū- mėjimų metu lyginant su remisija (p<0,05), bet remisijos metu lyginant IL- 8 periferiniame kraujyje ir skrepliuose su sveikais asmenimis – skirtumo negauta. Tiriant CRB ir kumuliaciniu rūkymo intensyvumą sąsajų nerasta.

Nustatytas tiesioginis ryšys tarp migravusių periferinio kraujo neut- rofilų ir indukuotų skreplių neutrofilų skaičiau. IL-8 koncentracija tiesiogiai įtakojo migravusių periferinio kraujo neutrofilų skaičių visose LOPL ser- gančiųjų grupėse. Sveikų asmenų IL-8 ir migravusių neutrofilų sąsajų nerasta.

Išvados:

1. Padidėjęs periferinio kraujo neutrofilų ir monocitų bei indukuotų skrep- lių neutrofilų ir makrofagų skaičius yra susijęs su pablogėjusia plaučių funkcija ir rūkymo intensyvumu bakterinės ir nebakterinės lėtinės obs- trukcinės plaučių ligos paūmėjimo metu.

2. Indukuotų skreplių neutrofilų ir makrofagų apoptozė bei fagocitozė susilpnėja, o reaktyvių deguonies formų susidarymas padidėja labiau bakterinio lėtinės obstrukcinės plaučių ligos metu lyginant su nebakteriniu paūmėjimu.

3. Nustatytas pakitęs periferinio kraujo neutrofilų ir monocitų aktyvumas bakterinės kilmės lėtinės obstrukcinės plaučių ligos metu lyginant su nebakterinės kilmės paūmėjimu – susilpnėjusi neutrofilų ir monocitų apoptozė ir fagocitozė, sustiprėjęs chemotaksis bei padidėjęs periferinio kraujo neutrofilų reaktyvių deguonies formų susidarymas.

4. Bakterinės ir nebakterinės kilmės lėtinės obstrukcinės plaučių ligos paūmėjimo metu padidėjusi interleukino-8 koncentracija susijusi su su- stiprėjusiu periferinio kraujo neutrofilų chemotaksiu, o C reaktyviojo baltymo padidėjusi koncentracija nesusijusi su plaučių funkcija ir rū- kymo intensyvumu.

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CURRICULUM VITAE (CV)

First name: Mindaugas

Surname: Vaitkus

Address: Department of Pulmonology and Immunology,

Lithuanian University of Health Sciences Eiveniu str. 2, LT-50009, Kaunas Lithuania Phone: +37037326895 +37067610168 Fax: +37037337504 e-mail: vaitkus.mindaugas@gmail.com Medical Education:

1998–2004 Studies at the Faculty of Medicine, Kaunas University of Medicine

2004–2005 Residency of general practice, Clinic Hospital of Panevėžys

2005–2009 Residency of Pulmonology, Hospital of Kaunas University of Medicine

Current positions:

2009–2014 Doctoral studies in Biology, Kaunas University of Medicine, Kaunas, Lithuania

Membership of professional societies

Lithuanian Society of Pulmonology and Allergy European Respiratory Society ERS

Knowledge of languages

Lithuanian, English, Russian