Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function

Cytokine 50 (2010) 152–157

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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function Olaf Eickmeier a,*, Marisa Huebner b, Eva Herrmann c, Ulrich Zissler a, Martin Rosewich a, Patrick C. Baer d, Roland Buhl b, Sabina Schmitt-Grohé e, Stefan Zielen a, Ralf Schubert a a

Department of Pediatric Pulmonology, Allergy and Cystic Fibrosis, Children’s Hospital, Goethe-University, Frankfurt, Germany Pulmonary Department, Internal Medicine, University Hospital Mainz, Germany Institute of Biostatistics and Mathematical Modeling, Goethe-University, Frankfurt, Germany d Department of Internal Medicine III, Goethe-University, Frankfurt, Germany e Children’s Hospital Medical Center, University of Bonn, Germany b c

a r t i c l e

i n f o

Article history: Received 9 October 2009 Received in revised form 26 January 2010 Accepted 1 February 2010

Keywords: Cystic fibrosis COPD Cytometric bead array Biomarkers Inflammation

a b s t r a c t Lung diseases like cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are associated with chronic airway inflammation. The aim of our study was to compare a complex biomarker profile in order to characterize specific inflammatory patterns in sputum of patients with CF and COPD. Induced sputum samples of 19 CF-, 26 COPD patients and 21 healthy controls were analyzed for concentrations of IL-1b, IL-2, IL-6, IL-8, IL-13, IP-10, MCP-1, IFN-c and TNF-a using the new cytometric bead array (CBA) technology. Significant differences in airway biomarker profiles of CF and COPD were detected. Patients with CF showed a significant increase in IL-1b, IL-6, IL-8, IL-13, TNF-a, IFN-c and MCP-1. COPD patients showed an increase in IL-6, IL-8, IL-13 and MCP-1 compared to healthy controls. CF and COPD compared to each other exhibited differences in IL-1b, IL-2, IL-8, TNF-a, IFN-c and MCP-1 levels. Significant correlations between the parameters of lung function and sputum biomarker levels were found. Analyzing induced sputum allows characterization of specific airway biomarker profiles in CF and COPD and can be related to the clinical status of the patient. CBA of induced sputum seems to be a pivotal tool to characterize pulmonary inflammation. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are chronic airway diseases [1,2] with different causes that share some features in pathologic changes and clinical presentation. In these diseases, there are chronic mucosal and airway inflammation, each with distinct pathophysiologic features, but a

Abbreviations: BSA, bovine serum albumin; CBA, cytometric bead array; CCL, chemokine (C–C motif) ligand; CF, cystic fibrosis; COPD, chronical obstructive pulmonary disease; CXCL, chemokine (C–X–C motif) Ligand; DTT, dithiotreitol; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; IFN-c, interferonc; IL, interleukin; IP-10, interferon-inducible protein-10; MCP-1, monocyte chemoattractant protein-1; lg, micrograms; MIP-1a, macrophage inflammatory protein-1 a; ml, milli-liters; PBS, phosphate buffered saline; P., Pseudomonas; RANTES, regulated on activation normal T cell expressed and secreted; TNF-a, tumor necrosis factor a; TH1, T helper 1; TH2, T helper 2. * Corresponding author. Address: Department of Pediatric Pulmonology, Allergy and Cystic fibrosis, Children’s Hospital, Goethe-University, Theodor-Stern-Kai 7, D60590 Frankfurt/Main, Germany. Tel.: +49 69 6301 83063; fax: +49 69 6301 83349. E-mail address: [email protected] (O. Eickmeier). 1043-4666/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2010.02.004

common increase in the infiltration of neutrophils [3] in addition to a variety of inflammatory mediators [4]. The pathologic processes in these diseases seem to involve progressive inflammatory responses with elements of tissue remodeling [5], airway obstruction and reduction in expiratory flow rates [6]. In COPD, an abnormal inflammatory response of the lung to noxious particles or gases is present [7]. Although cigarette smoking is the main pathological driver of COPD [8,9], other factors may be involved. Genetic predisposition could explain why only a proportion of cigarette smokers develop COPD [10]. Other factors include particulates in environmental pollution and exposure to biomass combustion, which may explain why some patients who develop COPD are never-smokers [11]. In CF, the inflammatory response is driven mainly by bacterial infections, especially Pseudomonas species, which leads to tissue breakdown and severe lung damage [12]. Markers of inflammatory activity play an important role for assessment and management of those respiratory diseases [13]. Due to technical reasons, previous investigations of sputum in chronic lung diseases, like CF and COPD, focused on cytology and determination of single inflammatory mediators [14–16].

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The determination of cytokines and chemokines in induced sputum by cytometric bead array (CBA) promises to be of substantial benefit for sputum investigations because numerous analytes can be measured simultaneously with a small sample volume [17]. In addition to this, CBA is faster and more cost effective than ELISA technology. In our study, CBA was used to determine sputum levels of proinflammatory cytokines and chemokines (IL-1b, IL-2, IL-6, IL-8, IL13, IP-10, INF-c, MCP-1 and TNF-a) in induced sputum of patients with CF and COPD in comparison to healthy control subjects. We hypothesized, that the analysis of these different pleiotropic inflammatory mediators may lead to pathognomonic patterns for the underlying disease. Furthermore, it was postulated that these patterns of biomarkers show a significant correlation to the clinical status (lung function) of our patients. 2. Materials and methods 2.1. Subjects and selection Patients were recruited from the Division of Pediatric Pulmonology, Allergy and Cystic fibrosis, Johann Wolfgang Goethe-University Hospital, Frankfurt, Germany and the Department of Internal Medicine, Johannes Gutenberg-University Hospital, Mainz, Germany. The population of this study consisted of 19 clinically stable patients with cystic fibrosis (18 were Pseudomonas aeruginosa-infected), 26 clinically stable patients with COPD and 21 non-smoking healthy control subjects. Clinical stability was defined as absence of acute exacerbation of disease 6 weeks prior to inclusion. Acute exacerbation was defined by two of the following symptoms: Fever >38.0 °C, increase of sputum, significant increase of C-reactive Protein (CrP) and significant weight loss. Exclusion criteria were absence of current use of systemic antibiotic and steroid treatment, clinically relevant renal, cardiac, or hepatic (ALT/AST >3 times of the upper normal limit, portal hypertension) dysfunction, chronic gastrointestinal disease not related to CF and pregnancy. The clinical characteristics of our study subjects are summarized in Table 1. 2.2. Sample collection and processing Subjects first inhaled 200 lg Salbutamol and subsequent nebulised hypertonic saline at concentrations of 3%, 4% and 5% for every 7 min. This bronchial stimulus caused expectoration of sputum. Before inhalation of hypertonic saline, the mouth was cleaned by flushing with water. Sputum was processed within 2 h of collection [18]. The selected sputum plugs were as far as possible without saliva, processed into a weighed Eppendorf tube and processed with 2 wt/vol of 0.1% Dithiothreitol (DTT). Afterwards 4 wt/

Table 1 Patient characteristics.

Age (years) Sex (f/m) FVC (% pred.) FEV1 (% pred.) Ps. a. S. a. Other

Healthy controls (n = 21)

CF (n = 19)

COPD (n = 26)

27 (20–42) 6:15 104 (87–122) 106 (88–122) – – –

27 (10–40) 12:7 87 (58–123) 67 (38–119) 18 6 15

64 (46–79) 7:15 83 (51–131) 51 (27–83) – – –

f, female; m, male; FVC (% pred.), Forced Vital Capacity in % predicted; FEV 1 (% pred.), Forced Expiratory Volume in 1 s in % predicted; Ps. a., Pseudomonas aeruginosa colonization, S. a., Staphylococcus aureus colonization, other: Achromobacter xylosoxidans (2), Aspergillus fumigatus (1), Candida albicans (4), Enterobacter cloacae (2), Moraxella catarrhalis (1), Proteus vulgaris (1), Serratia marcescens (2), Streptococcus (2).

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vol of PBS was added. Samples were filtered through 48 lm mesh and centrifuged for 10 min at 790g to remove the cells. Supernatants were stored at 80 °C until further analysis. 2.3. Cytometric bead array Concentrations of nine different cytokines/chemokines were determined in sputum samples using the BD™ CBA Flex Set System for the measurement of IL-1b, IL-2, IL-6, IL-8, IL-13, IP-10, INF-c, MCP-1 and TNF-a (BD Bioscience-PharMingen, San Diego, CA, USA) levels. Each BD™ CBA Flex Set contained one bead population with distinct fluorescence intensity, as well as the appropriate phycoerythrin (PE) detection reagent and standard. The tests were performed according to the manufacturer’s advice, and samples were run in duplicate [17,19]. For analyses of the cytokines/chemokines, we added the same concentration of DTT (0.025%) as in the sputum supernatant to the standard curve and enzyme immunoassay buffer. Interassay average coefficients of variation (%CV) of the test are: IL-1b, 6.68; IL-2, 7.16; IL-6, 7.13; IL-8, 11.92; IL-13, 11.23; IFN-c, 8.20; IP-10, 13.23; MCP-1, 6.81; TNF-a, 13.01. The lower limits of detection of the cytokines/chemokines are: IL-1b, 2.3 pg/ml; IL-2, 11.2 pg/ml; IL-6, 1.6 pg/ml; IL-8, 1.2 pg/ml; IL-13, 0.6 pg/ml; IFN-c, 1.8 pg/ml; IP-10, 0.5 pg/ml; MCP-1, 1.3 pg/ml; TNF-a, 0.7 pg/ml. 2.4. Pulmonary function test Pulmonary function tests were performed according to American Thoracic Society guidelines for performance and acceptance prior to sputum-induction [20]. 2.5. Statistical analysis Differences between the groups were performed by the nonparametric Kruskal–Wallis Test. Correlations between biomarkers and clinical parameters were verified by using Spearman correlation coefficients (r). Logistic regression analysis was performed to assess combinations of biomarkers with the different patient groups. A probability of p < 0.05 was regarded as significant (SPSS statistical program, version 17; SPSS Inc., Chicago, IL, USA). In Fig. 1, data are shown as individual values and median. In the text, data are given as median and range. 3. Results All nine biomarkers were detectable in nearly every sputum sample derived from patients with CF and COPD and compared to healthy control subjects. However, looking at IP-10, MCP-1, IL2, IFN-c and IL-13 some sputum samples were under the limit of detection. Measured parameters were grouped in neutrophilic inflammatory cytokines (Fig. 1A: IL-1b, IL-6 and TNF-a), chemokines (Fig. 1B: IL-8, IP-10 and MCP-1) and TH1/TH2 -cytokines (Fig. 1C: IL-2, IFN-c and IL-13). The inflammatory cytokines/chemokines IL-1b, IL-6, IL-8 and TNF-a from CF patients (IL-1b: 3279 pg/ml, 0–18532, p < 0.001; IL-6: 147 pg/ml, 82.4–930; p < 0.001; IL-8: 49683 pg/ml, 14757– 112687, p < 0.001; TNF-a: 179 pg/ml, 56.1–2377, p < 0.001) and IL-6 and IL-8 from patients with COPD (IL-6: 119 pg/ml, 0.0– 2993, p < 0.001; IL-8: 4257 pg/ml, 674–29965, p < 0.01) were significantly increased compared to controls (IL-1b: 62.4 pg/ml, 0.0– 398; IL-6: 25.5 pg/ml, 0.0–886; IL-8: 739 pg/ml, 228–16923; TNFa: 0.0 pg/ml, 0.0–68.9). IL-1b (p < 0.01), IL-8 (p < 0.001) and TNFa (p < 0.001) were also significantly increased in CF compared to COPD. The TH1 cytokine, IL-2, was decreased in CF (0.0 pg/ml, 0.0–51.4, p < 0.01), whereas IFN-c was significantly elevated in

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Fig. 1. Levels of biomarkers in CF, COPD and healthy controls in induced sputum. The biomarkers were grouped in (A) inflammatory cytokines, (B) inflammatory chemokines and (C) TH1 and TH2 representative cytokines. Determination of biomarkers were performed by CBA. Group differences among CF, COPD and healthy controls were analyzed by the nonparametric Kruskal–Wallis Test. Data presented by single values and median. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. Patterns of lung function and sputum biomarkers in CF, COPD and healthy controls. Lung function data and levels of sputum biomarkers are displayed as a twodimensional pseudo-color representation. The horizontal axis shows the analyzed parameter. The color bar along the right vertical axis shows the concentration of the analyzed parameter increasing from dark blue to red. Patients and controls are sorted by increasing age from bottom to top cells in each group. White cells indicate missing data.

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CF-sputum (122 pg/ml, 78.8–306, p < 0.001) compared to controls (IL-2: 36.8 pg/ml, 0.0–228; IFN-c: 26.1 pg/ml, 0.0–181). IFN-c together with IL-8 also enables an exact identification of CF patients in this study. IFN-c (pg/ml) + 0.00525  IL-8 (pg/ml) was larger than 190 in all patients with CF but smaller than 190 in all patients with COPD and all controls. IL-2 was decreased in CF (0 pg/ml, 0– 51, p < 0.001) compared to COPD, whereas IFN-c was significantly increased in CF (122 pg/ml, 78.8–306) compared to COPD (35.0 pg/ ml, 15–123, p < 0.001). The TH2 cytokine, IL-13, was significantly increased in CF (33.2 pg/ml, 0.0–52.9, p < 0.05) and COPD patients (0.0 pg/ml, 0–129, p < 0.05) compared to the healthy control group (0.0 pg/ml, 0.0–74.7). The chemokine, IP-10, showed no significant differences between the groups. MCP-1 was increased in CF (307 pg/ml, 158–3309, p < 0.001) and COPD sputum (151 pg/ml, 0–709, p < 0.05) compared to controls (83.5 pg/ml, 0–793), and in addition, it was also significantly increased in CF (0 pg/ml, 79– 306) compared to COPD patients (0 pg/ml, 0–129, p < 0.05). (Figs. 1A–C and 2.) Moreover, we correlated clinical lung function data (FEV1 and FVC) to cytokine/chemokine sputum levels. CF-sputum levels of IL-1b, IL-6, IL-8, IFN-c, TNF-a and MCP-1 showed a significant negative correlation with FEV1 and FVC. IL-2 levels of our CF patients showed a significant positive correlation with FEV1 and FVC. Concerning our COPD group, IL-6, IL-8 and MCP demonstrated a significant negative correlation with FEV1 and FVC. FEV1 was especially low in the COPD group. All COPD patients had FEV1 below 85% whereas all controls had FEV1 above 85%. IP-10 showed a negative correlation with FVC in this group (Table 2, Fig. 2). 4. Discussion Markers of inflammatory activity are important for the assessment and management of chronic respiratory diseases like CF and COPD [21]. In order to study the local inflammatory response non-invasively, we employed a standardized and validated sputum-induction protocol [18]. Numerous studies have analyzed inflammatory cytokines in sputum, in relation to clinical status [22–25]. The new CBA technique allows characterization of specific

Table 2 Correlations between parameters of lung function and cytokines/chemokines in induced sputum of patients with CF and COPD.

CF

COPD

Parameters of lung function

Mediators

FEV1 FEV1 FEV1 FEV1 FEV1 FEV1 FEV1

IL-1b Il-2 IL-6 IL-8 IFN-c MCP-1 TNF-a

r 0.48 0.60 0.60 0.64 0.59 0.58 0.63

p-Value 0.005 0.001 0.001 0.001 0.001 0.001 0.001

FVC FVC FVC FVC FVC FVC FVC

IL-1b Il-2 IL-6 IL-8 IFN-c MCP-1 TNF-a

0.36 0.52 0.58 0.54 0.54 0.53 0.57

0.039 0.002 0.001 0.001 0.001 0.002 0.001

FEV1 FEV1 FEV1

IL-6 IL-8 MCP-1

0.47 0.50 0.34

0.002 0.001 0.005

FVC FVC FVC FVC

IL-6 IL-8 IP-10 MCP-1

0.52 0.33 0.32 0.48

0.001 0.041 0.045 0.002

FEV1, Forced Expiratory Volume in 1 s in % predicted; FVC, Forced Vital Capacity in % predicted; r, correlations between disease biomarkers and FEV1 and FVC were analyzed using Spearman correlation coefficients (r).

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airway biomarker profiles from small sputum samples in order to investigate a broad range of inflammatory parameters simultaneously. Interestingly, all nine biomarkers were detectable by CBA technology in sputum from patients with CF, COPD and also in our healthy control subjects. We hypothesized that the analysis of these different cytokines and chemokines may lead to pathognomonic patterns for the underlying disease. Elevated inflammatory sputum and serum mediators like IL-6, IL-8 and TNF-a and a negative correlation to disease severity have been previously reported in CF and COPD patients [26–31]. As expected, the elevation of IL-8 was most pronounced in CF (p < 0.001), followed by COPD (p < 0.01) compared to healthy controls. Both diseases are characterized by an enormous neutrophilic enhanced inflammation. The pro-inflammatory cytokine, IL-6, was also significantly elevated in CF (p < 0.001) and COPD (p < 0.001) compared to controls. IL-6 is well known for its B-Cell stimulating activity [32]. Although it is produced by different cells, e.g., activated T-lymphocytes, fibroblasts and mast cells, the transcription of the IL-6 gene is regulated by bacterial endotoxin (LPS) and pro-inflammatory cytokines like IL-1 and TNF-a [33]. TNF-a was significantly elevated in CF compared to COPD (p < 0.001) and controls (p < 0.001). TNF-a is produced primarily by macrophages and other mononuclear phagocytes and has many functions in the development of inflammation and the activation of other leukocytes [34,35]. IL-1b has wide-ranging functions that prepare the body to combat disease. Notably, only in our CF patients IL-1b was significantly elevated. IL-1 is a major mediator of inflammation, performing numerous functions related to host defense mechanisms by regulating not only the immune system but also the neuronal and endocrine systems [36]. The most important function of IL-1 is the induction of fever. One alternative effect of IL-1 is suppression of appetite, which is a well known symptom of CF patients. In summary, when focussing on the pro-inflammatory cytokines, the CF patients showed extremely high levels of inflammatory cytokines. IL-1b and TNF-a levels were distinctive for CF, compared to COPD patients. Next, we analyzed representative mediators of the TH1 and TH2 system. In CF, the TH1 response that is characterized by the production of IL-2 and IFN-c may predominate, as showed in patients with chronic P. aeruginosa lung infection [37]. Accordingly, IFN-c was significantly increased in CF compared to COPD (p < 0.001) and controls (p < 0.001). In contrast to previous observations, IL-2 was significantly reduced in our CF group (p < 0.001) in comparison to COPD and healthy controls. Moreover, IL-2 CF-sputum levels showed a positive correlation to FEV1 and FVC. Accordingly, it was found that IL-2 levels are significantly higher in CF- P. aeruginosa- negative patients compared to levels in P. aeruginosa-infected patients [38]. Our CF group consisted mainly (95%) of P. aeruginosainfected patients. This provides evidence, that pulmonary IL-2 downregulation in CF might represent an appropriate effort of the immune system to defend against inflammation triggered by P. aeruginosa. As a representative marker for a TH2 driven inflammation, we analyzed sputum IL-13 levels. IL-13, which shares a receptor component and signaling pathways with IL-4, acts on B-Cells to produce IgE. Leukocyte transmigration into the airways is orchestrated by cytokines like IL-4, IL-5 and IL-13, and coordinated by specific chemokines like eotaxin and RANTES (CCL5), in combination with adhesion molecules, such as VCAM-1 and E-selectin [39,40]. IL-13 is by far the most potent inducer of eotaxin expression in airway epithelial cells [26]. Moreover, IL-13 alters both ciliated cell differentiation and ciliary beat frequency, while it increases the production of secretory cells [41]. In our study, IL13 was significantly increased in CF (p < 0.05) and COPD (p < 0.05), indicating an ongoing TH2 mediated inflammation.

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The chemokine, MCP-1, was significantly increased in CF compared to COPD (p < 0.05) and controls (p < 0.001). Accordingly, it was shown that alveolar macrophages and CC chemokines (MIP3a (CCL20), MCP-1 (CCL2), MIP-1a (CCL-3) and MIP-1b (CCL4)) were elevated in the lungs of young children with CF, even in the absence of pulmonary infection [42]. The CXC chemokine, IP-10 (CXCL10), which is said to have a central role in TH1-mediated diseases [43], showed no significant differences between the groups. Furthermore, we found significant correlations between sputum biomarker levels and clinical status, represented by FEV1 and FVC (Table 2). This study is reporting a panel of correlation estimates between lung function parameters and sputum cytokines/chemokines levels in CF and COPD, in order to develop future disease related anti-inflammatory intervention strategies. IL-6, IL-8 and MCP-1 were found to be significant negatively correlated to lung function in CF and COPD. IL-1b, IFN-c and TNF-a were found to be negatively correlated to lung function in CF only. Former studies correlating single sputum biomarkers such as IL-8 with lung function parameters showed comparable results with our study [14,22,23,44]. In conclusion, CF and COPD share a common elevation of inflammatory biomarkers. Beside the common inflammatory markers, IL-1b and TNF-a, we found significantly higher sputum levels of IL-8 and IFN-c in CF, which points to a pivotal role of IL8 as a chemoattractant and a TH1 driven inflammation in the pathogenesis of CF. IFN-c together with IL-8 enables an exact identification of CF patients in this study. IFN-c (pg/ml) + 0.00525  IL-8 (pg/ml) was larger than 190 in all patients with CF but smaller than 190 in all patients with COPD and all controls. Our data suggests that sputum IL-8 and IFN-c levels might be a critical measure in subjects with cystic fibrosis. We recommend that a CF lung inflammatory biomarker panel include a combination of IL-8 and IFN-c in clinical trials, but also in a routine clinical setting in order to characterize the individual inflammatory response. Induced sputum is relatively non-invasive, reliable and a valid technique to measure airway inflammation [45]. It can be applied even in the more severe conditions and can be used repeatedly. Therefore, we conclude that the assessment of induced sputum is a valid method of assessing airway diseases. We showed that cytokine patterns help to differentiate inflammatory airway diseases from healthy subjects. More important than the measurement of single cytokine or chemokine levels is the analysis of biomarker profiles (Fig. 2) to determine the severity of lung inflammation. Furthermore, the data support the role of sputum biomarkers as a mirror of disease severity in chronic airway diseases like CF and COPD. The development of accurate, non-invasive measures of pulmonary inflammation would allow the evaluation of the extent to which existing anti-inflammatory drugs may ameliorate inflammation in CF and COPD [46]. This study showed that analyzing sputum mediators by CBA seems to be a pivotal tool to characterize pulmonary inflammation.

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