Serum amyloid A (SAA) in induced sputum of asthmatics: A new look to an old marker

International Immunopharmacology 6 (2006) 1569 – 1576 www.elsevier.com/locate/intimp

Serum amyloid A (SAA) in induced sputum of asthmatics: A new look to an old marker Ferhan Ozseker a,⁎, Suna Buyukozturk a , Bilge Depboylu b , Dilek Yilmazbayhan c , Ebru Karayigit c , Aslı Gelincik a , Sema Genc b , Bahattin Colakoglu a , Murat Dal a , Halim Issever d a

Istanbul University, Istanbul Faculty of Medicine, Department of Internal Medicine, Division of Allergy, Turkey b Istanbul University, Istanbul Faculty of Medicine, Department of Biochemistry, Turkey c Istanbul University, Istanbul Faculty of Medicine, Department of Pathology, Turkey d Istanbul University, Istanbul Faculty of Medicine, Department Biostatistics, Turkey Received 12 April 2006; received in revised form 26 May 2006; accepted 30 May 2006

Abstract Background: Some cellular and soluble markers of inflammation in induced sputum have been used for studying airway inflammation in asthma. The aim of this study was to assess the usefulness of systemic inflammation marker serum amyloid A (SAA) in blood and induced sputum to monitor the airway inflammation in asthmatic patients. Method: Seventeen non-smokers newly diagnosed mild to moderate asthmatic patients and 10 healthy volunteers were included in this prospective parallel designed study. Inflammatory cell counts, SAA and eosinophil cationic protein (ECP) levels were measured in sera and induced sputum of both groups. All tests were repeated in the asthma group after 6 months of inhaled steroid therapy. The diagnostic accuracy and reproducibility of sputum and blood SAA were estimated. Results: Serum and induced sputum SAA and ECP levels, sputum eosinophils and neutrophils of untreated asthmatic patients were significantly greater compared to the control group. Sputum and sera SAA levels and sputum neutrophils remained unchanged after the 6 months of anti-inflammatory therapy, although ECP levels, sputum eosinophils and macrophages were significantly reduced. The area under the curve (AUC) for sputum SAA was found equal to AUC for sputum ECP (0.87). The reproducibility of sputum SAA was satisfactory (ICC = 0.84) as well. Conclusion: Our findings suggest that systemic inflammatory marker SAA may be used as a reliable inflammatory marker in asthma. The facts that whether it remarks an ongoing inflammation unresponsive to treatment in the airways or reflects a systemic inflammation needs to be clarified with further studies. © 2006 Elsevier B.V. All rights reserved. Keywords: Serum amyloid A; ECP; Induced sputum; Acute phase proteins; Eosinophil

⁎ Corresponding author. Feyzullah Mah. Şehit Hikmetalp Cad. Okurlar Sitesi, D Blok, No:10, Maltepe 34843, Istanbul, Turkey. Tel.: +90 2163713124. E-mail addresses: [email protected] (F. Ozseker), [email protected] (S. Buyukozturk), [email protected] (B. Depboylu), [email protected] (D. Yilmazbayhan), [email protected] (E. Karayigit), [email protected] (A. Gelincik), [email protected] (S. Genc), [email protected] (B. Colakoglu), [email protected] (M. Dal), [email protected] (H. Issever). 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.05.006

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1. Introduction Asthma is an inflammatory disorder of lower airways. The severity of illness is determined by the degree of inflammation [1]. Bronchial biopsy specimens reveal that bronchial mucosa of asthmatics are rich from eosinophilic infiltrate and bronchoalveolar lavage fluids has a high proportion of eosinophils and some eosinophil derived mediators [eosinophil cationic protein (ECP), etc.] [2,3]. In recent years, measurement of inflammatory markers in induced sputum has been accepted as valid, responsive and reproducible [3–5]. The ECP levels in BAL fluid and sputum has been the most frequently used marker to estimate the severity of bronchial inflammation in asthmatics [6]. Serum amyloid A (SAA) is an acute phase protein whose level is elevated in the blood during infection, trauma, surgery, burns, tissue infarction, inflammation, neoplasia and stress [7]. SAA production is induced mainly by IL-6, IL-1 and TNF-α, that are multifunctional cytokines produced by many cell types of body [7,8]. SAA has been considered to be produced by hepatocytes and secreted into serum. However, UrieliShoval et al. [9] demonstrated that SAAmRNA is expressed in normal human epithelial component of many organs and cells. SAA could be released locally in some organ-specific diseases. For example, expression of SAAmRNA and protein had been found in human atherosclerotic lesions [10]. In some disorders, the SAA levels have prognostic importance. High levels of SAA in cystic fibrosis indicate antibiotic resistant Pseudomonas aeroginosa infection [11], while it emphasizes the risk of development of systemic amyloidosis in destructive lung tuberculosis [12]. In very few studies, a relation between the SAA and asthma was emphasized. Jousilahti et al. [13] revealed that there was a positive correlation between SAA and asthma prevalence and they concluded that systemic inflammation also existed besides the local inflammation in bronchial asthma. Büyüköztürk et al. [14] demonstrated that blood SAA concentrations were higher in patients with asthma and allergic rhinitis. The aim of the current study was to evaluate whether SAA levels increase in both induced sputum and blood of asthmatics and to find out if it could be used as an inflammatory marker of asthmatic inflammation. We considered only SAA among the other acute phase reactants because it is involved in many aspects of pathogenesis of many inflammatory disorders. To document the usefulness of this marker in asthma, the validity and reproducibility of these measurements were estimated. The blood and sputum ECP levels, which

have been accepted as established markers demonstrating the degree of airway inflammation in asthma, was selected as comparative index to determine the criterion validity of SAA [15]. 2. Methods 2.1. Study design and the patients This study is a prospective investigation with a parallel design. Twenty non-smoker patients with untreated mild to moderate persistent asthma and 12 non-smoker healthy subjects were involved. The Ethical Committee of Istanbul University Faculty of Medicine approved the study protocol, and informed written consent was obtained from each participant. 2.2. Inclusion criteria for the patients The patients between 18 and 60 years of age having asthmatics symptoms (recurrent dyspnea, cough and/or wheeze attacks, nocturnal dyspnea, exercise induced dyspnea and/or cough and/or wheeze) for at least 6 months and proven asthma diagnosis according to GINA guidelines [16] and never been treated with inhaled or systemic steroids, leukotrien antagonists and long acting beta-2 agonists were involved into the study. 2.2.1. Inclusion criteria for the control subjects The volunteers who had no allergies, any symptoms or diseases related with respiratory tract, without any immunological disorder were included. 2.2.2. Exclusion criteria for the patients and control subjects The patients and controls who did not meet the inclusion criteria, who were unable to cooperate (in keeping appointments and drug using), the ones that have had viral/bacterial upper or lower respiratory infections for the last 6 weeks and the pregnant women were excluded from the study. 2.3. Diagnostic procedures All of the patients with the complaints suggesting asthma underwent physical examination and allergy skin prick tests with 25 common inhalant allergens (Vivodiagnost, ALK Benelux BV, Groningen, the Netherlands). A skin prick test was considered as positive if the diameter of wheal was 3 mm larger than negative control. Pulmonary function tests were performed (Vmax 20c, SensoriMedics, California, USA). The patients whose forced expiratory volume at 1st second (FEV1) were <80% of predicted with an increase of at least 12% 15 min after the inhalation of 200 μg salbutamol were accepted as having reversible lung function. Values were expressed as a percentage of the predicted value. The patients whose FEV1s were equal or more than 80% of predicted underwent bronchial provocation tests with

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2.6. Treatment

methacoline. Asthma was diagnosed in the patients with a suggestive history for asthma plus reversibility in lung function test or PC20 value under the 4 mg/mL in methacholine provocation test [16]. For the evaluation of blood inflammatory markers, 10 mL of peripheral venous blood samples were driven from all of the participants. In asthmatics, peripheral eosinophils were counted at once. Collected sera after the coagulation of blood for 1 h at room temperature were stored at −70 °C until the analysis for ECP and SAA. At the same day, induced sputum was obtained from the patients and control subjects using a previously described method [17,18].

The asthmatic patients were treated with fluticason propionate 250–500 μg b.i.d. (Flixotide™, Diskus GlaxoWellcome Operations, UK) and short acting β2-agonists (Ventolin™, Glaxo-SmithKline Operations, UK) as needed. The patients were asked to visit the clinic at the 3rd and 6th months of the treatment. At the third month visit, adherence of therapy was evaluated and physical examination was done; at the final visit in the 6th month, pulmonary function tests, blood and sputum analysis were repeated beside physical examination.

2.4. Sputum induction and processing

2.7. Statistical analysis

Sputum was induced by protocol based on that described by Pin et al. [18]. Ten minutes after 200 μg salbutamol inhalation, hypertonic saline was nebulized using an ultrasonic nebulizer (Ultraneb 2000, Somerset, USA) for 5min periods up to 30 min. To elicit adequate sputum amount, the concentration was increased at intervals of 7 min from 3% to 4% to 5%. The FEV1 was recorded every 10 min during the nebulization. If the FEV1 fell by more than 10% of the post-bronchodilator value, the concentration of hypertonic saline was not increased. If the FEV1 fell by more than 20% of the post-bronchodilator value, nebulization was discontinued. The sputum sample was expectorated into a conical polycarbonate tube and eight-fold volume phosphate buffered saline (PBS) was added to disperse the sputum [19,20]. It was shown that some mediator levels were affected by dithiothreitol (DTT) during the sputum processing [19–21]. In this study, DTT was not used because it is not known how it affects SAA levels in sputum. For the separation of the mucous part, the sputum was filtrated through 4 mm nylon gauze. The filtrate was then centrifuged at 790×g for 10 min. The precipitate was examined for viability and cell counts by cytospin (Shandon Cytospin 3, USA); the fluid part was put into an eppendorf tube and stored at − 70 °C until analysis of the markers. Cytospin preparations were prepared and stained with May-Grünwald/ Giemsa (BDH Laboratory Supplies), and 400 non-squamous cells were counted per slide.

According to the type of distribution, Student's t-test or Wilcoxon signed ranks test was used to compare the values between pre- and post-treatment paired samples of the asthmatic group. Mann–Whitney's U-test was performed to compare the values between the asthma and control groups. Correlations between the variables were examined using Spearman's correlation analysis. We determined the diagnostic accuracy of sputum and blood SAA comparing with that of ECP by generating a receiver-operating characteristic (ROC) curve for each test. The areas under the curves (AUCs) were compared as described [22,23]. In addition, reproducibility of sputum and serum SAA was estimated by the concordance correlation coefficient and the Bland-Altman correlation coefficient [24]. 3. Results Three patients among asthmatics and two subjects among control group were excluded because of being not able to give adequate and good quality sputum. Seventeen asthmatic patients and 10 healthy volunteers were considered suitable for the evaluation. The demographic data of the study groups was shown in Table 1. Table 1 The clinical description of the cases and comparison of the parameters between pre-treatment and post-treatment asthmatics and pretreatment asthmatics versus control cases

2.5. Biochemical analysis hSAA kits were purchased from BioSource Europe S.A. Nivelles, Belgium. The concentrations of SAA (in ng/mL) in the serum and thawed supernatant were determined by solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (ELX 800, Biotech Instrument Inc., Cortland, New York, USA). The lower limit of detection was 5 ng/mL for hSAA in serum. The human ECP kit was purchased from ©Pharmacia Diagnostics AB, Uppsala, Sweden. Serum and sputum supernatant hECP levels were measured by the ImmunoCap method (UniCAP 100, Uppsala, Sweden) and the detection limit was < 0.5 μg/L.

BT asthma patients (n = 17)

PT asthma patients (n = 10)

Control (n = 10)

Sex (male/ female) Age (years) BMI FEV1 (%)

(2/15)

(1/9)

(3/7)

35.5 ± 8.6 26.6 ± 7.3 72.8 ± 12.2

30.0 ± 8.5 – 100.1 ± 12.7

31.0 ± 14.6 26.3 ± 8.3 104.5 ± 10.5

FEV1/FVC (%)

75.7 ± 7.1

97.0 ± 10.1

98.0 ± 10.5

P⁎,⁎⁎

0.769⁎ 0.726⁎ 0.000⁎, 0.000⁎⁎ 0.002⁎, 0.000⁎⁎

BT: before treatment, PT: post-treatment. ⁎Comparison between pre-treatment patients and controls; ⁎⁎pretreatment versus post-treatment asthmatics.

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Control blood and sputum examinations after 6 months could be taken from only 10 patients because two patients had become pregnant, two had to quit treatment, two could not give adequate sputum and one patient was abroad. Asthma was mild persistent in four patients and moderate persistent in 13. The body mass index was not correlated with any inflammatory marker. Skin prick tests for one or more aeroallergens were positive in eight patients. Any correlations were not found between the skin test results and inflammatory markers in serum and sputum. The %FEV1 values were significantly increased after the inhaled steroid therapy (Table 1). 3.1. Sputum cell counts Sputum eosinophils and neutrophils of the asthmatics were significantly higher than those of the control patients (Table 2). After the treatment, sputum eosinophils and macrophages were strikingly decreased while neutrophils remained unchanged. Sputum eosinophils were negatively correlated with FEV1 and positively correlated with blood eosinophils, serum SAA, sputum SAA and macrophages (Table 3). Table 2 Inflammatory cells and soluble markers in blood and induced sputum BT asthma patients (n = 17)

PT asthma patients (n = 10)

Control (n = 10)

P⁎,⁎⁎

Blood SAA (ng/mL)

18.8 ± 8.5

20.7 ± 9.7

12.2 ± 5.9

ECP (μg/L)

10.7 ± 9.13

2.8 ± 1.4

5.3 ± 1.6

0.038⁎, 0.862⁎⁎ 0.029⁎, 0.011⁎⁎

Eosinophil (× 103/μL)

3.16 ± 2.57

ND

ND

Sputum Total cell count (× 103/mL) Squamous cell (%)

13.0 ± 12.2

9.5 ± 3.4

5.5 ± 5.0

33.1 ± 7.8

36.7 ± 3.8

39.7 ± 5.9

Eosinophil (%)

11.3 ± 7.34

5.1 ± 2.5

1.5 ± 1.5

Macrophage (%)

10.6 ± 6.7

4.8 ± 1.4

19.4 ± 8.1

Neutrophil (%)

20.4 ± 3.8

21.8 ± 3.2

15.3 ± 2.7

Lymphocyte (%)

11.3 ± 4.2

17.9 ± 5.3

12.4 ± 3.9

Ciliated epithelial cell (%) SAA (ng/mL)

14.1 ± 7.1

14.3 ± 2.3

11.7 ± 3.6

11.3 ± 3.8

12.9 ± 4.9

6.2 ± 2.9

ECP (μg/L)

35.8 ± 49.7

2.1 ± 0.4

2.8 ± 1.6

Table 3 Correlations between markers in blood and sputum and clinical measurements

Blood SAA (ng/mL) Sputum SAA (ng/mL) Sputum eosinophils (%) Blood eosinophils (× 103/μL)

FEV1 (L/min)

Blood ECP (μg/L)

Sputum ECP (μg/L)

NS

NS

0.402⁎⁎ 0.558⁎

− 0.506⁎ NS

Sputum Sputum eosinophil neutrophil (%) (%) NS

0.481⁎⁎

NS

− 0.554⁎ 0.588⁎ 0.761⁎

–

NS

0.454⁎⁎

0.775⁎

NS

NS

0.732⁎ 0.761⁎

Only significant correlation figures were given. ⁎⁎p < 0.05; ⁎p < 0.01; NS = not significant.

3.2. SAA and ECP levels Sputum and blood SAA values of the asthmatics were significantly higher than those of the control group. No change was found in either sputum or blood SAA levels after the treatment (Figs. 1 and 2). There was negative correlation between sputum SAA and FEV1 and positive correlation between sputum SAA and blood eosinophils (Table 3). Before the anti-inflammatory treatment, the sputum and serum ECP levels of the patients were significantly higher compared to the control group. The ECP levels in both sputum and blood significantly decreased after the treatment (Fig. 3). There were positive correlations between sputum/serum ECP levels and sputum eosinophil counts (Figs. 4 and 5).

0.385⁎, 0.036⁎⁎ 0.021⁎, 0.194⁎⁎ 0.000⁎, 0.031⁎⁎ 0.011⁎, 0.018⁎⁎ 0.001⁎, 0.185⁎⁎ 0.528⁎, 0.005⁎⁎ 0.552⁎, 0.280⁎⁎ 0.001⁎, 0.205⁎⁎ 0.015⁎, 0.025⁎⁎

ND: not done, BT: before treatment, PT: post-treatment. ⁎Comparison between pre-treatment patients and controls; ⁎⁎pretreatment versus post-treatment asthmatics.

Fig. 1. Sputum SAA was significantly higher in the asthma group compared to the control group (p = 0.001) and did not change after the anti-inflammatory treatment of 6 months (p = 0.205).

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Fig. 2. Blood SAA concentrations were also significantly increased compared to control group (p = 0.038) and remain high despite therapy (p = 0.862).

In the ROC curves, AUC for sputum SAA was greater (0.87) than AUC for serum SAA (0.72) (Table 4). On the other hand, sputum SAA and ECP had similar AUCs (both 0.87), while sputum eosinophil (0,99) was the most sensitive parameter (Fig. 6). The cut-off values obtained giving equal weight to sensitivity and specificity for the serum and sputum SAA were as follows respectively: 12.53 ng/mL (sensitivity 76%, specificity 80%) and 9.6 ng/mL (sensitivity 71%, specificity 90%). As the SAA values did not change after the

Fig. 3. Blood and sputum ECP concentrations of the patient and control groups. The differences between the pre-treatment values of the patients and control subjects were significant as well as the values between the pre- and post-treatment concentrations of the patients.

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Fig. 4. Correlation between blood ECP concentrations and sputum eosinophils in asthmatic subjects.

treatment, reproducibility of SAA measurement in induced sputum was calculated between these two measurements and interclass correlation coefficient (ICC) was found 0.84 (satisfactory) (Fig. 7).

4. Discussion We found that systemic inflammation marker SAA was significantly increased in the blood and sputum of

Fig. 5. Correlation between sputum ECP concentrations and sputum eosinophils in asthmatic subjects.

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Table 4 The results of the ROC plot for sputum and blood parameters Parameters

AUC Std. Asymptotic 95% (area under error significance Confidence the curve) interval

Sputum ECP Sputum SAA Sputum Eosinophil Serum ECP Serum SAA

0.87 0.87 0.99 0.77 0.72

Lower Upper 0.07 0.07 0.10 0.09 0.10

0.001 0.001 0.000 0.02 0.05

0.735 0.734 0.975 0.585 0.530

1.006 1.007 1.013 0.957 0.923

asthmatics and was unaffected by the anti-inflammatory treatment. The results of the current study seem compatible with the findings of previous two studies about SAA and asthma [13,14] and, to our knowledge, this is the first study evaluating the SAA levels in induced sputum. The pathogenetic role of airway inflammation in asthma has been demonstrated in a number of investigations. Many different cells of various types including eosinophils, neutrophils, macrophages, monocytes, etc. have been emphasized to take part in that inflammation via producing different mediators. A number of mediators released from macrophages that play an important role in asthmatic inflammation are implied for their involvement either in the beginning or persisting of inflammation [25,26]. IL-1β, IL-6, IL-8, platelet activating factor (PAF) and tumour necrosing factor α (TNFα) are markers released from macrophages and stimulate many inflammatory cells as well as the production of acute phase proteins [7,9,26]. This stimulation might be the cause of the augmentation of well known acute phase reactant SAA in bronchial

Fig. 6. The ROC plot for sputum ECP, SAA and eosinophils. Area under the curves (AUCs) for SAA and ECP were equal (0.87); however, sputum eosinophil count was more sensitive than these markers (0.99).

Fig. 7. Blant-Altman test results for the comparison of the two sputum SAA measurements. Mean test score was 11.75 ± 4.04 and difference score was − 2.81 ± 3.76. The standard deviation of the difference scores was observed to scatter in ±2 S.D. Pearson correlation coefficient between the two measurements was 0.776 (p = 0.008) and interclass correlation coefficient (ICC) was 0.84 (p = 0.0047, 95% CI: 0.39– 0.96).

tissue. High blood SAA levels suggested us that SAA might be synthesized from some other sources as well as the airways, which might support the belief that there is a systemic inflammation in asthma. As another surprising result, the SAA levels were not affected by the antiinflammatory treatment of six months. Accordingly, we thought that chronic airway inflammation in asthma might be lasting despite clinical improvements, as it had been emphasized by many histological studies. ECP, which has been proposed as one of the most important markers to estimate the severity of airway inflammation in asthmatics [18], was also found as a highly sensitive and specific parameter in this current study. The mean ECP values in our study were less than those found in previous studies. The reason of this was the absence of DTT in sputum processing. However, sputum and serum ECP levels of the patients were higher than those of the control group and significantly decreased after the anti-inflammatory treatment. Positive correlations between sputum and serum ECP levels with sputum eosinophil counts were found. Using ROC curve, we found that sputum eosinophils were more accurate than both serum and sputum ECP as compatible to Pizzichini's [3] study. Comparing to the ECP ROC curves, sputum SAA was observed as an accurate marker as sputum ECP while serum SAA had less accuracy. In this current study, eosinophils and neutrophils in the asthmatic patients were detected to be much higher than controls. In spite of that we used only PBS in

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sputum processing, our sputum cell counts in asthmatic patients were compatible with inflammation pattern of asthma. Efthimiadis et al. [19] had demonstrated that DTT-treated versus only PBS-treated sputum displayed similar cell counts except the cell viability which was higher in PBS-treated samples. For that reason, our sputum processing technique seems to be valid and accurate. In most studies, the high sputum neutrophils had been proposed to be usually found in severe and uncontrolled asthma. On the other hand, it is well known that neutrophilic airway inflammation usually might be seen during acute attacks or viral infections in asthmatic patients [27,28]. It was surprising that we detected high neutrophil counts beside increased eosinophil counts in the induced sputum of our patients which were clinically diagnosed as mild to moderate asthma. Gibson et al. [29] demonstrated elevated neutrophil counts in induced sputum of non-eosinophilic persistent asthmatic patients. In that study and some others, neutrophil counts had been found unaffected by inhaled steroid treatment [27,29]. We also observed that high neutrophil proportion persisted in sputum of asthmatic patients after the anti-inflammatory treatment. This finding suggested that neutrophils play an important role in the airway inflammation even in mild and moderate asthma and might remain high despite the clinical improvement. Considering that the SAA was the other biological marker that persisted to be elevated after the anti-inflammatory therapy, we might speculate that SAA and neutrophils might be in relation with each other. In conclusion, the results of the current study suggest that systemic inflammatory marker SAA in induced sputum may reflect the inflammatory status in asthma and may be used to monitor this inflammation. As a result, whether SAA could remark a systemic inflammation needs to be clarified with further studies. References [1] Louis R, Lau LCK, Bron AO, Roldaan AC, Radermecker M, Djukanovic R. The relationship between inflammation and asthma severity. Am J Respir Crit Care Med 2000;161:9–16. [2] Bousquet J, Chanez P, Lacoste JY, Barneon G, Ghavanian N, Enander I, et al. Eosinophilic inflammation in asthma. N Engl J Med 1990;323:1033–9. [3] Pizzichini E, Pizzichini MM, Efthimiadis A, Dolovich J, Hargreave FE. Measuring airway inflammation in asthma: eosinophils and eosinophilic cationic protein in induced sputum compared with peripheral blood. J Allergy Clin Immunol 1997;99:539–44. [4] Fahy JV, Boushey HA, Lazarus SC, Mauger EA, Cherniack RM, Chinchilli VM, et al. Safety and reproducibility of sputum induction in asthmatic subjects in a multicenter study. Am J Respir Crit Care Med 2001;163:1470–5.

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