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Pentraxin 3 sputum levels differ in patients with chronic obstructive pulmonary disease vs asthma
Ann Allergy Asthma Immunol xxx (2015) 1e5
Contents lists available at ScienceDirect
Pentraxin 3 sputum levels differ in patients with chronic obstructive pulmonary disease vs asthma Fabiano L. Schwingel, MD, PhD; Emilio Pizzichini, MD, PhD; Tulia Kleveston, MD; Edelton F. Morato, MD, PhD; José T. Pinheiro, MSc; Leila J.M. Steidle, MD, PhD; Felipe Dal-Pizzol, MD, PhD; Cristiane C. Rocha, MSc; and Marcia M.M. Pizzichini, MD, PhD NUPAIVA, Federal University of Santa Catarina, Florianópolis, Brazil
A R T I C L E
I N F O
Article history: Received for publication July 21, 2015. Received in revised form September 21, 2015. Accepted for publication October 2, 2015.
A B S T R A C T
Background: Immune response has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD) and asthma. Pentraxin 3 (PTX3) is a multifunctional pattern recognition protein and an important component of the innate immune system that can be assessed in blood and induced sputum. Objective: To determine whether PTX3 measured in induced sputum could discriminate patients with COPD from patients with asthma. Methods: A cross-sectional study of 68 participants (27 with COPD, 25 with asthma, and 16 healthy controls) was performed. At study inclusion sputum was collected and total and differential cell numbers and PTX3 levels were determined. Results: Pentraxin 3 was detected in 89% of patients with COPD, 56% of patients with asthma, and 19% of controls (P ¼ .001). It discriminated participants with COPD (24.6 ng/mL, 0e384 ng/mL) from controls (0 ng/ mL, 0e36 ng/mL, P < .001) and from participants with asthma (1.2 ng/mL, 0e100 ng/mL, P ¼ .01; area under the receiver operating curve 0.82 [0.71e0.94]). Regression analyses determined that sputum PTX3 and neutrophil counts were independently associated with COPD. In addition, PTX3 levels were independently associated with COPD severity. Conclusion: Pentraxin 3 sputum levels are increased in patients with COPD and has good power to discriminate these patients from patients with asthma and healthy individuals. Ó 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Introduction Asthma and chronic obstructive pulmonary disease (COPD) are the most prevalent chronic airway diseases, characterized by airflow obstruction and inflammation.1,2 Although appearing distinct in typical presentations, these diseases might share common functional and inflammatory features, depending on a specific phenotype, and might coexist.3,4 Most asthma cases are characterized by reversible airflow limitation, airway hyperresponsiveness, and T-helper cell type 2 (TH2) eosinophilic airway inflammation, whereas COPD is predominantly characterized by irreversible airflow obstruction, TH1 neutrophilic airway inflammation, and chronic bacterial colonization.
Reprints: Marcia M.M. Pizzichini, MD, PhD, Division of Internal Medicine, Universidade Federal de Santa Catarina, NUPAIVAeHospital Universitário, Campus UniversitárioeTrindade, 88040-970 Florianópolis, SC, Brazil; E-mail: mpizzich@ matrix.com.br. Disclosures: Authors have nothing to disclose. Funding Sources: The authors have received funding from CNPq and FAPESC.
The innate immune response, which is the first line of defense against pathogens, plays a key role in the initiation, activation, and orientation of adaptive immunity. In body fluids, innate responses are characterized by the presence of collectins, ficolins, complement components, and pentraxins. Pentraxin 3 (PTX3) is a protein originating from the pentraxin family, which includes C-reactive protein.5 Despite these molecules exerting similar actions in the regulation of the inflammatory response, many differences exist between C-reactive protein and PTX3, including gene organization, protein oligomerization, and expression pattern.5 For example, Creactive protein is produced in the liver in response to interleukin (IL)-6, whereas mainly inflammatory cells produce PTX3 in response to IL-15. Although originally identified as a cytokine-inducible gene from endothelial cells and fibroblasts, other cells are known to express PTX3 at exposure to inflammatory stimuli. Several cells can produce PTX3, but its major source includes dendritic cells and macrophages.6 Furthermore, neutrophils can store PTX3, which is secreted with neutrophil extracellular traps.7 In addition, it is not expressed in lymphocytes, eosinophils, and basophils.
http://dx.doi.org/10.1016/j.anai.2015.10.003 1081-1206/Ó 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
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F.L. Schwingel et al. / Ann Allergy Asthma Immunol xxx (2015) 1e5
Therefore, it is reasonable to presume that PTX3 could be a specific biomarker of innate responses and thus could correspondingly be a relative specific biomarker of COPD. Few studies have investigated PTX3 levels in patients with COPD and have failed to demonstrate significant differences in pulmonary, sputum, and plasma PTX3 expression among never-smokers, smokers without COPD, and patients with COPD.8,9 However, PTX3 levels have been strongly correlated with the number of neutrophils in sputum. Furthermore, the role of PTX3 sputum levels in differentiating COPD from asthma has yet to be evaluated. In the present study, the authors hypothesized that PTX3 would be increased in the sputum of patients with COPD compared with those with asthma and control subjects. In addition, they investigated whether PTX3 levels in sputum in the fluid phase was correlated with COPD severity. Thus, a cross-sectional study was performed to determine PTX3 levels in the sputum of normal subjects, patients with asthma, and patients with COPD and the correlation of its levels with COPD severity.
Methods Design and Participants This was a cross-sectional study. Data were obtained from 68 participants who attended the respiratory medicine clinic at the authors’ university. Patients with COPD were included if they had a clinical diagnosis of smoking-related COPD confirmed by a postbronchodilator ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity lower than 0.70 (n ¼ 27). Participants with asthma (n ¼ 25) were included if they had previously been diagnosed with asthma, which was confirmed by a ratio of FEV1 to forced vital capacity lower than 0.80 and an improvement in FEV1 of at least 12% was observed after inhaled salbutamol spray at the dose of 400 mg. Patients with asthma were nonsmokers or exsmokers (<10 pack-years). Healthy controls (n ¼ 16) were nonsmoking adults who had no nasal or chest symptoms, current or previous respiratory problems, asthma or allergic rhinitis, or occupational exposure to dust, chemical products, or fumes, and had normal spirometry and normal methacholine airway responsiveness (provocative concentration of methacholine causing a 20% decrease in FEV1 >8 mg/mL). Treatment at enrollment was recorded. Inhaled corticosteroid doses were expressed as beclomethasone equivalents. Atopy was defined by at least 1 skin prick test with a reaction wheal larger than 3 mm compared with the control test reaction. Exclusion criteria included a current or recent (past month) respiratory tract infection, exacerbation of respiratory disease, or a course on oral steroids or antibiotics in the previous month. The research committee of the Federal University of Santa Catarina approved the study and all subjects gave written informed consent.
Measurements Subject characteristics were documented in a structured questionnaire. Spirometry was performed using a Koko-Trek Spirometer (Trudell Medical, London, Ontario, Canada) before and 20 minutes after the inhalation of 400 mg of salbutamol through a powder metered-dose inhaler and aerochamber spacer.10 Reference values were taken from a study by Crapo et al.11 Sputum induction was performed as described by Pizzichini et al12,13 using a Medix ultrasonic nebulizer (Canadian Medical Products, Ltd, Markham, Ontario, Canada) with an output of 0.9 mL/min. Sputum was separated from the expectorate and examined as described by Pizzichini et al.14 Total cell count of nonsquamous cells was obtained in a modified Neubauer hemocytometer, with cell viability determined by trypan blue exclusion. The sputum was labeled
Table 1 Clinical and functional characteristics of subjects Asthma (n ¼ 25) Age (y), median (minemax) Women, n (%) Atopic, n (%) Smokers/ ex-smokers, n Smoker (pack-years), median (minemax) Pulmonary drugs On ICS, n (%) On LABA, n (%) ICS dose (mg/da), median (minemax) Pre-BD FEV1 (%) Post-BD FEV1 (%) Pre-BD FEV1/FVC (%) Post-BD FEV1/FVC (%) DFEV1 post-BD (L) DFEV1 post-BD (%)
COPD (n ¼ 27)
39 (18e68)
62 (52e77)
20 (80) 18 (72) 0/8
6 (21) 15 (56) 5/22
Control (n ¼ 16)
P value
31 (20e59) <.001b,c 9 (56) 7 (44) 0/0
<.001c, .051b .003d .003b
53.4 (20e132)
0
<.001b,c
8 (32) 12 (41) 9 (36) 16 (59) 466 (0e2934) 533 (0e3260)
0 0 0
.01b <.001b, .036d .1d
1.4 (0e10)
66.8 77.9 65.2 71.1 0.36 18.8
(18.2) (16.9) (11.7) (11.8) (0.24) (12.8)
47.8 51.9 47.7 47.5 0.12 9.9
(16.7) (17.1) (12.5) (12.5) (0.09) (7.8)
94.6 97.5 84.6 87.1 0.09 3.2
(11.9) (11.2) (5.2) (4.5) (0.1) (4.3)
<.001b,c,d .01d, <.001b,c <.001b,c,d <.001b,c,d <.001c,d .004c, <.001d
Abbreviations: BD, bronchodilator; COPD, chronic obstructive pulmonary disease; D, difference; FEV1, forced expiratory volume in first second; FVC, forced vital capacity; ICS, inhaled corticosteroid; LABA, long-acting b2-agonist; max, maximum; min, minimum. a Equivalent to beclomethasone. b COPD vs control. c Asthma vs COPD. d Asthma vs control.
eosinophilic if sputum eosinophils constituted at least 3.0% and neutrophilic if sputum neutrophils constituted at least 50.0%. PTX3 Enzyme-Linked Immunosorbent Assay Pentraxin 3 enzyme-linked immunosorbent assay was performed with a DuoSet enzyme-linked immunosorbent assay development kit (R&D, Minneapolis, Minnesota) according to the manufacturer’s instructions. Absorbance values were read after 15 minutes at 405 nm in an automatic enzyme-linked immunosorbent assay reader. Intra- and inter-assay coefficients of variation were less than 10%. The lower detection limit of the assay was 0.15 ng/mL. Statistical Analysis Standard descriptive statistics were used to characterize the study population. Continuous variables are presented as median and interquartile interval or mean and SD depending on their distribution. Differences in baseline characteristics and biomarker levels were tested using the c2 or Mann-Whitney rank-sum U test, as appropriate. The correlation between biomarkers was carried out using the Spearman coefficient correlation test. Receiver operating characteristic curves were constructed by plotting sensitivity vs 1 specificity, and the area under the receiver operating characteristics curve was used to evaluate the ability of each biomarker level to discriminate subjects with from those without COPD. Linearity between continuous variables and the dependent variable was demonstrated using locally weighted scatter plot smoothing. In this way, PTX3 concentrations required a logarithmic transformation to satisfy the linearity assumption. Multivariate logistic regression analyses were performed to examine the association among PTX3, sputum cellularity, and COPD (as the dependent variable). Markers yielding P values less than .2 by univariate analysis were imputed in forward multivariate logistic regression analysis. Thus, 1 model was constructed that included sputum PTX3 levels and absolute macrophage, neutrophil,
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Table 2 Cellular characteristics and pentraxin 3 levels in sputum Asthma (n ¼ 25) TCC 106/mL 4.9 (1.8e12.3) Neutrophils (%) 32.4 (2.5e84.4) Eosinophils (%) 11.7 (0e60) Macrophage (%) 47.9 (14.3e83.5) Lymphocytes (%) 3.1 (0e10.5) PTX3 (ng/mL) 1.2 (0e100) PTX3 above limit 14 (56) of detection
COPD (n ¼ 27) 10.6 58.8 2.3 35.3 1.4 24.6 24
Control (n ¼ 16)
P value
(1.5e32.1) 3.4 (0.4e6) <.001a, .004b (7.0e85.3) 23 (3e72) <.001a, .01b (0e18) 0.16 (0e1) .003c, .003b (11.7e87) 66 (22e94) .02c, <.001a (0e4) 3.6 (0.5e10.5) .012b, .01a (0e384) 0 (0e36) .009a, .012b (89) 3 (19) <.001a,b
Abbreviations: COPD, chronic obstructive pulmonary disease; PTX3, pentraxin 3; TCC, total cell count. a COPD vs controls. b Asthma vs COPD. c Asthma vs controls.
and eosinophil counts. Linear regression analyses were performed to examine the association between biomarkers and clinical variables and the decrease of %FEV1 (as the dependent variable) in patients with COPD. Variables significantly correlated with %FEV1 were imputed in linear regression analysis. Thus, 1 model was constructed that included sputum PTX3 levels, sputum neutrophil counts, age in years, and pack-years. Statistical significance was defined as a P value less than .05. Data were analyzed using SPSS 18.0 for Windows (SPSS, Inc, Chicago, Illinois). Results Patient Description As expected, patients with COPD were older and had significantly lower lung function compared with patients with asthma and healthy controls (Table 1). Smoking history was significantly more frequent in patients with COPD. COPD severity was classified as mild (stage I, n ¼ 1 [4%]), moderate (stage II, n ¼ 14 [52%]), severe (stage III, n ¼ 10 [37%]), or very severe (stage IV, n ¼ 2 [7%]) according to criteria of the Global Initiative for Chronic Obstructive Lung Disease.2 Sputum Biomarkers and COPD Diagnosis Compared with patients with asthma, healthy controls and patients with COPD had significantly increased total cell count and proportion of neutrophils (Table 2). As expected, subjects with asthma had larger proportions of eosinophils than patients with COPD and healthy control subjects (Table 2).
Figure 2. Induced sputum pentraxin 3 (iPTX3) levels in patients with chronic obstructive pulmonary disease (COPD) and patients with asthma with or without sputum neutrophilia (asthma with neutrophilia, n ¼ 7; COPD with neutrophilia, n ¼ 17).
Pentraxin 3 was detected in 88.9% of patients with COPD, 56.0% of patients with asthma, and 18.8% of controls (P ¼ .001; Table 2). It discriminated patients with COPD (24.6 ng/mL, 0e384 ng/mL) from controls (0 ng/mL, 0e36 ng/mL, P < .001) and from patients with asthma (1.2 ng/mL, 0e100 ng/mL, P ¼ .01; Fig 1), with an area under the receiver operating characteristics curve of 0.82 (0.71e0.94). PTX3 levels were significantly higher in participants with COPD and sputum neutrophilia (28.4 ng/mL, 11.3e304 ng/mL) compared with those without neutrophilia (11.9 ng/mL, 0e90.8 ng/mL, P ¼ .008; Fig 2). The discriminative value of sputum neutrophil levels was 0.81 (0.71e0.91). In patients with asthma and sputum neutrophilia, PTX3 levels were similar to those without neutrophilia (1.2 ng/mL [0e82 ng/mL] vs 0 ng/mL [0e14.3 ng/mL], respectively, P ¼ .6; Fig 2). PTX3 levels were not affected by the presence or absence of eosinophilia (data not shown). Regression analyses were performed to determine any independent association between biomarkers and COPD. Only sputum PTX3 and neutrophil counts were independently associated with COPD. Sputum Biomarkers and COPD Severity Because PTX3 was independently associated with COPD, the authors investigated whether some of these biomarkers were related to COPD severity (assessed by post-bronchodilator FEV1%). In a linear regression model, only pack-years (standardized b ¼ 0.436 [0.550 to 0.029], P ¼ .031) and PTX3 (standardized b ¼ 0.443 [5.78 to 0.37], P ¼ .028) were independently related to COPD severity. Discussion
Figure 1. Induced sputum pentraxin 3 (iPTX3) levels in patients with chronic obstructive pulmonary disease (COPD), patients with asthma, and healthy individuals.
In the present study, patients with COPD had significantly higher levels of PTX3 in induced sputum fluid phases than did those with asthma or healthy controls. In addition, PTX3 sputum levels were associated with sputum neutrophilia and inversely correlated with the magnitude of airflow obstruction only in patients with COPD. These findings suggest that PTX3 is a potential marker of airway inflammation in COPD. Despite great interest in the role of PTX3 in several inflammatory diseases, little is known about its role in respiratory diseases, including asthma and COPD.15e18 This understanding has been
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limited by conflicting results from the few clinical studies on this subject. Hamon et al15 found higher levels of PTX3 in the sputum of subjects with COPD compared with those with cystic fibrosis. In contrast, Van Pottelberge et al8 did not find any significant increase in PTX3 levels in the lungs of patients with COPD. An increase in PTX3 immunoreactivity has been observed in bronchial tissues of patients with allergic asthma compared with healthy controls.18 In these patients, PTX3 was localized mainly in the smooth muscle but was not related to the severity of the disease.18 In patients with COPD, but not in those with heart failure, plasma PTX3 levels were correlated with IL-1b levels.16 These dissimilarities between studies could reflect several aspects of PTX3 production, such as the presence of infection, site of production, measurement, and cells involved in its production. The present results support the role of PTX3 in sputum levels as a marker of COPD and its severity. Pentraxin 3 levels are almost undetectable in the blood and tissues of healthy individuals and increases rapidly in response to inflammatory stimuli in different pathologies, such as autoimmune diseases, septic shock,19 acute myocardial infarction,20e22 and traumatic brain injury.23 PTX3 knockout mice respond equally to lipopolysaccharide as wild-type controls.24 In contrast, mice that overexpress PTX3 are protected against sepsis induced by cecal ligation, perforation, and lipopolysaccharide.7 In the present study, all patients were free from a recent exacerbation and infection (in the 4 weeks before the study), but induced sputum fluid phase levels of PTX3 were significantly higher in the COPD group than in the asthma group. These results support a truly differential effect of COPD-related inflammation and it is not as a result of an enhanced PTX3 secretion in response to infection. However, these results cannot exclude that the PTX3 high levels seen in patients with COPD are related to local microbiomes and further studies are required to investigate this possibility. Nevertheless, one could presume that the presence of PTX3 indicates a greater degree of lung inflammation. In this context, PTX3 is highly specific to macrophages and neutrophils, and the present study showed that it is relatively specific for COPD-associated lung inflammation compared with patients with asthma and normal subjects. The issue of PTX3 being expressed in the lungs is not new and many experimental studies support this notion. For instance, lung epithelial cells stimulated by tumor necrosis factor-a (TNF-a) induced PTX3 production in human and mice.23,25 Also, PTX3 protein was found expressed constitutively by human airway smooth muscle cells and significantly upregulated by TNF-a and IL-1b, but not by TH2 (IL-4, IL-9, IL-13), TH1 (interferon-g), or TH17 (IL-17) cytokines.25 After exposure to TNF-a, nuclear factor-kB interacts with the human PTX3 promoter, at least in fibroblasts26 and epithelial cells.27 Furthermore, in lung epithelial cells stimulated by TNF-a, the inhibition of c-Jun N-terminal kinase pathway decreased PTX3 expression.27 The results of clinical studies are discrepant, which might be due to differences in study populations, disease severity, and methods. The present study has some limitations. First, the cross-sectional design is insufficient to infer causality (eg, PTX3 induces inflammation in patients with COPD). Second, the study design does not allow the determination of the temporal evolution of PTX3 levels and disease progression. Third, PTX3 levels in the sputum, and not in the lung, were investigated; thus, the authors cannot ascertain whether PTX3 is really produced by neutrophils, although it is very likely. Despite these limitations, this approach is clinically relevant and could add useful information to COPD diagnosis and probably to asthmaeCOPD overlap syndrome. Fourth, the authors cannot exclude the effect of treatment on PTX3 sputum levels because approximately half the patients were using inhaled corticosteroids or a long-acting b2-agonist. The control subjects and patients with
asthma were significantly younger than the patients with COPD. Some age-related diseases can increase PTX3 levels (eg, coronary artery disease), and aging is associated with increased inflammation. This fact might have led to a misinterpretation of the results. However, because PTX3 levels were measured in the sputum and PTX3 is produced by inflammatory cells (and not, for example, by the liver), it was expected that the sputum variation of PTX3 levels would be related mainly to airway inflammation and not to systemic inflammation or aging. In addition, only a weak correlation was observed between age and PTX3 in the present sample (r ¼ 0.36). Furthermore, the regression model did not alter the association between PTX3 and COPD (age was inclusive). In conclusion, the present study showed that PTX3 in sputum levels was significantly increased in patients with COPD and showed a good discriminatory power distinguishing these patients from patients with asthma and healthy individuals. These results open the perspective of using PTX3 in future studies to better characterize overlap syndrome.
References [1] Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA) 2015. Available from: http://www.ginasthma.org. Accessed September 30, 2015. [2] Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2015. Available from: http://www.goldcopd.org/. Accessed September 30, 2015. [3] de Marco R, Pesce G, Marcon A, et al. The coexistence of asthma and chronic obstructive pulmonary disease (COPD): prevalence and risk factors in young, middle-aged and elderly people from the general population. PLoS One. 2013; 8:e62985. [4] Hardin M, Cho M, McDonald ML, et al. The clinical and genetic features of COPDeasthma overlap syndrome. Eur Respir J. 2014;44:341e350. [5] Alles VV, Bottazzi B, Peri G, Golay J, Introna M, Mantovani A. Inducible expression of PTX3, a new member of the pentraxin family, in human mononuclear phagocytes. Blood. 1994;84:3483e3493. [6] Introna M, Alles VV, Castellano M, et al. Cloning of mouse ptx3, a new member of the pentraxin gene family expressed at extrahepatic sites. Blood. 1996;87:1862e1872. [7] Daigo K, Hamakubo T. Host-protective effect of circulating pentraxin 3 (PTX3) and complex formation with neutrophil extracellular traps. Front Immunol. 2012;3:378. [8] Van Pottelberge GR, Bracke KR, Pauwels NS, Vermassen FE, Joos GF, Brusselle GG. COPD is associated with reduced pulmonary interstitial expression of pentraxin-3. Eur Respir J. 2012;39:830e838. [9] Mantovani A. Pentraxin-3 in COPD: innocent bystander or amplifier? Eur Respir J. 2012;39:795e796. [10] Miller MR, Hankinson J, Brusasco V, et al. Standardisation of spirometry. Eur Respir J. 2005;26:319e338. [11] Crapo RO, Morris AH, Gardner RM. Reference spirometric values using techniques and equipment that meet ATS recommendations. Am Rev Respir Dis. 1981;123:659e664. [12] Pizzichini E, Pizzichini MM, Efthimiadis A, et al. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med. 1996;154:308e317. [13] Pizzichini E, Pizzichini MM, Leigh R, Djukanovic R, Sterk PJ. Safety of sputum induction. Eur Respir J Suppl. 2002;37:9se18s. [14] Pizzichini E, Pizzichini MM, Efthimiadis A, Hargreave FE, Dolovich J. Measurement of inflammatory indices in induced sputum: effects of selection of sputum to minimize salivary contamination. Eur Respir J. 1996;9:1174e1180. [15] Hamon Y, Jaillon S, Person C, et al. Proteolytic cleavage of the long pentraxin PTX3 in the airways of cystic fibrosis patients. Innate Immun. 2013;19:611e622. [16] Beghe B, Verduri A, Bottazzi B, et al. Echocardiography, spirometry, and systemic acute-phase inflammatory proteins in smokers with COPD or CHF: an observational study. PLoS One. 2013;8:e80166. [17] Rovere P, Peri G, Fazzini F, et al. The long pentraxin PTX3 binds to apoptotic cells and regulates their clearance by antigen-presenting dendritic cells. Blood. 2000;96:4300e4306. [18] Garlanda C, Bottazzi B, Bastone A, Mantovani A. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu Rev Immunol. 2005;23:337e366. [19] Muller B, Peri G, Doni A, et al. Circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients. Crit Care Med. 2001;29:1404e1407. [20] Peri G, Introna M, Corradi D, et al. PTX3, A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans. Circulation. 2000; 102:636e641.
F.L. Schwingel et al. / Ann Allergy Asthma Immunol xxx (2015) 1e5 [21] Salio M, Chimenti S, De Angelis N, et al. Cardioprotective function of the long pentraxin PTX3 in acute myocardial infarction. Circulation. 2008;117: 1055e1064. [22] Latini R, Maggioni AP, Peri G, et al. Prognostic significance of the long pentraxin PTX3 in acute myocardial infarction. Circulation. 2004;110:2349e2354. [23] Gullo Jda S, Bertotti MM, Silva CC, et al. Hospital mortality of patients with severe traumatic brain injury is associated with serum PTX3 levels. Neurocrit Care. 2011;14:194e199. [24] Han B, Haitsma JJ, Zhang Y, et al. Long pentraxin PTX3 deficiency worsens LPS-induced acute lung injury. Intensive Care Med. 2011;37:334e342.
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[25] Zhang J, Shan L, Koussih L, et al. Pentraxin 3 (PTX3) expression in allergic asthmatic airways: role in airway smooth muscle migration and chemokine production. PLoS One. 2012;7:e34965. [26] Basile A, Sica A, d’Aniello E, et al. Characterization of the promoter for the human long pentraxin PTX3. Role of NF-kappaB in tumor necrosis factor-alpha and interleukin-1beta regulation. J Biol Chem. 1997;272: 8172e8178. [27] Han B, Mura M, Andrade CF, et al. TNFalpha-induced long pentraxin PTX3 expression in human lung epithelial cells via JNK. J Immunol. 2005;175: 8303e8311.
Contents lists available at ScienceDirect
Pentraxin 3 sputum levels differ in patients with chronic obstructive pulmonary disease vs asthma Fabiano L. Schwingel, MD, PhD; Emilio Pizzichini, MD, PhD; Tulia Kleveston, MD; Edelton F. Morato, MD, PhD; José T. Pinheiro, MSc; Leila J.M. Steidle, MD, PhD; Felipe Dal-Pizzol, MD, PhD; Cristiane C. Rocha, MSc; and Marcia M.M. Pizzichini, MD, PhD NUPAIVA, Federal University of Santa Catarina, Florianópolis, Brazil
A R T I C L E
I N F O
Article history: Received for publication July 21, 2015. Received in revised form September 21, 2015. Accepted for publication October 2, 2015.
A B S T R A C T
Background: Immune response has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD) and asthma. Pentraxin 3 (PTX3) is a multifunctional pattern recognition protein and an important component of the innate immune system that can be assessed in blood and induced sputum. Objective: To determine whether PTX3 measured in induced sputum could discriminate patients with COPD from patients with asthma. Methods: A cross-sectional study of 68 participants (27 with COPD, 25 with asthma, and 16 healthy controls) was performed. At study inclusion sputum was collected and total and differential cell numbers and PTX3 levels were determined. Results: Pentraxin 3 was detected in 89% of patients with COPD, 56% of patients with asthma, and 19% of controls (P ¼ .001). It discriminated participants with COPD (24.6 ng/mL, 0e384 ng/mL) from controls (0 ng/ mL, 0e36 ng/mL, P < .001) and from participants with asthma (1.2 ng/mL, 0e100 ng/mL, P ¼ .01; area under the receiver operating curve 0.82 [0.71e0.94]). Regression analyses determined that sputum PTX3 and neutrophil counts were independently associated with COPD. In addition, PTX3 levels were independently associated with COPD severity. Conclusion: Pentraxin 3 sputum levels are increased in patients with COPD and has good power to discriminate these patients from patients with asthma and healthy individuals. Ó 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Introduction Asthma and chronic obstructive pulmonary disease (COPD) are the most prevalent chronic airway diseases, characterized by airflow obstruction and inflammation.1,2 Although appearing distinct in typical presentations, these diseases might share common functional and inflammatory features, depending on a specific phenotype, and might coexist.3,4 Most asthma cases are characterized by reversible airflow limitation, airway hyperresponsiveness, and T-helper cell type 2 (TH2) eosinophilic airway inflammation, whereas COPD is predominantly characterized by irreversible airflow obstruction, TH1 neutrophilic airway inflammation, and chronic bacterial colonization.
Reprints: Marcia M.M. Pizzichini, MD, PhD, Division of Internal Medicine, Universidade Federal de Santa Catarina, NUPAIVAeHospital Universitário, Campus UniversitárioeTrindade, 88040-970 Florianópolis, SC, Brazil; E-mail: mpizzich@ matrix.com.br. Disclosures: Authors have nothing to disclose. Funding Sources: The authors have received funding from CNPq and FAPESC.
The innate immune response, which is the first line of defense against pathogens, plays a key role in the initiation, activation, and orientation of adaptive immunity. In body fluids, innate responses are characterized by the presence of collectins, ficolins, complement components, and pentraxins. Pentraxin 3 (PTX3) is a protein originating from the pentraxin family, which includes C-reactive protein.5 Despite these molecules exerting similar actions in the regulation of the inflammatory response, many differences exist between C-reactive protein and PTX3, including gene organization, protein oligomerization, and expression pattern.5 For example, Creactive protein is produced in the liver in response to interleukin (IL)-6, whereas mainly inflammatory cells produce PTX3 in response to IL-15. Although originally identified as a cytokine-inducible gene from endothelial cells and fibroblasts, other cells are known to express PTX3 at exposure to inflammatory stimuli. Several cells can produce PTX3, but its major source includes dendritic cells and macrophages.6 Furthermore, neutrophils can store PTX3, which is secreted with neutrophil extracellular traps.7 In addition, it is not expressed in lymphocytes, eosinophils, and basophils.
http://dx.doi.org/10.1016/j.anai.2015.10.003 1081-1206/Ó 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
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F.L. Schwingel et al. / Ann Allergy Asthma Immunol xxx (2015) 1e5
Therefore, it is reasonable to presume that PTX3 could be a specific biomarker of innate responses and thus could correspondingly be a relative specific biomarker of COPD. Few studies have investigated PTX3 levels in patients with COPD and have failed to demonstrate significant differences in pulmonary, sputum, and plasma PTX3 expression among never-smokers, smokers without COPD, and patients with COPD.8,9 However, PTX3 levels have been strongly correlated with the number of neutrophils in sputum. Furthermore, the role of PTX3 sputum levels in differentiating COPD from asthma has yet to be evaluated. In the present study, the authors hypothesized that PTX3 would be increased in the sputum of patients with COPD compared with those with asthma and control subjects. In addition, they investigated whether PTX3 levels in sputum in the fluid phase was correlated with COPD severity. Thus, a cross-sectional study was performed to determine PTX3 levels in the sputum of normal subjects, patients with asthma, and patients with COPD and the correlation of its levels with COPD severity.
Methods Design and Participants This was a cross-sectional study. Data were obtained from 68 participants who attended the respiratory medicine clinic at the authors’ university. Patients with COPD were included if they had a clinical diagnosis of smoking-related COPD confirmed by a postbronchodilator ratio of forced expiratory volume in 1 second (FEV1) to forced vital capacity lower than 0.70 (n ¼ 27). Participants with asthma (n ¼ 25) were included if they had previously been diagnosed with asthma, which was confirmed by a ratio of FEV1 to forced vital capacity lower than 0.80 and an improvement in FEV1 of at least 12% was observed after inhaled salbutamol spray at the dose of 400 mg. Patients with asthma were nonsmokers or exsmokers (<10 pack-years). Healthy controls (n ¼ 16) were nonsmoking adults who had no nasal or chest symptoms, current or previous respiratory problems, asthma or allergic rhinitis, or occupational exposure to dust, chemical products, or fumes, and had normal spirometry and normal methacholine airway responsiveness (provocative concentration of methacholine causing a 20% decrease in FEV1 >8 mg/mL). Treatment at enrollment was recorded. Inhaled corticosteroid doses were expressed as beclomethasone equivalents. Atopy was defined by at least 1 skin prick test with a reaction wheal larger than 3 mm compared with the control test reaction. Exclusion criteria included a current or recent (past month) respiratory tract infection, exacerbation of respiratory disease, or a course on oral steroids or antibiotics in the previous month. The research committee of the Federal University of Santa Catarina approved the study and all subjects gave written informed consent.
Measurements Subject characteristics were documented in a structured questionnaire. Spirometry was performed using a Koko-Trek Spirometer (Trudell Medical, London, Ontario, Canada) before and 20 minutes after the inhalation of 400 mg of salbutamol through a powder metered-dose inhaler and aerochamber spacer.10 Reference values were taken from a study by Crapo et al.11 Sputum induction was performed as described by Pizzichini et al12,13 using a Medix ultrasonic nebulizer (Canadian Medical Products, Ltd, Markham, Ontario, Canada) with an output of 0.9 mL/min. Sputum was separated from the expectorate and examined as described by Pizzichini et al.14 Total cell count of nonsquamous cells was obtained in a modified Neubauer hemocytometer, with cell viability determined by trypan blue exclusion. The sputum was labeled
Table 1 Clinical and functional characteristics of subjects Asthma (n ¼ 25) Age (y), median (minemax) Women, n (%) Atopic, n (%) Smokers/ ex-smokers, n Smoker (pack-years), median (minemax) Pulmonary drugs On ICS, n (%) On LABA, n (%) ICS dose (mg/da), median (minemax) Pre-BD FEV1 (%) Post-BD FEV1 (%) Pre-BD FEV1/FVC (%) Post-BD FEV1/FVC (%) DFEV1 post-BD (L) DFEV1 post-BD (%)
COPD (n ¼ 27)
39 (18e68)
62 (52e77)
20 (80) 18 (72) 0/8
6 (21) 15 (56) 5/22
Control (n ¼ 16)
P value
31 (20e59) <.001b,c 9 (56) 7 (44) 0/0
<.001c, .051b .003d .003b
53.4 (20e132)
0
<.001b,c
8 (32) 12 (41) 9 (36) 16 (59) 466 (0e2934) 533 (0e3260)
0 0 0
.01b <.001b, .036d .1d
1.4 (0e10)
66.8 77.9 65.2 71.1 0.36 18.8
(18.2) (16.9) (11.7) (11.8) (0.24) (12.8)
47.8 51.9 47.7 47.5 0.12 9.9
(16.7) (17.1) (12.5) (12.5) (0.09) (7.8)
94.6 97.5 84.6 87.1 0.09 3.2
(11.9) (11.2) (5.2) (4.5) (0.1) (4.3)
<.001b,c,d .01d, <.001b,c <.001b,c,d <.001b,c,d <.001c,d .004c, <.001d
Abbreviations: BD, bronchodilator; COPD, chronic obstructive pulmonary disease; D, difference; FEV1, forced expiratory volume in first second; FVC, forced vital capacity; ICS, inhaled corticosteroid; LABA, long-acting b2-agonist; max, maximum; min, minimum. a Equivalent to beclomethasone. b COPD vs control. c Asthma vs COPD. d Asthma vs control.
eosinophilic if sputum eosinophils constituted at least 3.0% and neutrophilic if sputum neutrophils constituted at least 50.0%. PTX3 Enzyme-Linked Immunosorbent Assay Pentraxin 3 enzyme-linked immunosorbent assay was performed with a DuoSet enzyme-linked immunosorbent assay development kit (R&D, Minneapolis, Minnesota) according to the manufacturer’s instructions. Absorbance values were read after 15 minutes at 405 nm in an automatic enzyme-linked immunosorbent assay reader. Intra- and inter-assay coefficients of variation were less than 10%. The lower detection limit of the assay was 0.15 ng/mL. Statistical Analysis Standard descriptive statistics were used to characterize the study population. Continuous variables are presented as median and interquartile interval or mean and SD depending on their distribution. Differences in baseline characteristics and biomarker levels were tested using the c2 or Mann-Whitney rank-sum U test, as appropriate. The correlation between biomarkers was carried out using the Spearman coefficient correlation test. Receiver operating characteristic curves were constructed by plotting sensitivity vs 1 specificity, and the area under the receiver operating characteristics curve was used to evaluate the ability of each biomarker level to discriminate subjects with from those without COPD. Linearity between continuous variables and the dependent variable was demonstrated using locally weighted scatter plot smoothing. In this way, PTX3 concentrations required a logarithmic transformation to satisfy the linearity assumption. Multivariate logistic regression analyses were performed to examine the association among PTX3, sputum cellularity, and COPD (as the dependent variable). Markers yielding P values less than .2 by univariate analysis were imputed in forward multivariate logistic regression analysis. Thus, 1 model was constructed that included sputum PTX3 levels and absolute macrophage, neutrophil,
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Table 2 Cellular characteristics and pentraxin 3 levels in sputum Asthma (n ¼ 25) TCC 106/mL 4.9 (1.8e12.3) Neutrophils (%) 32.4 (2.5e84.4) Eosinophils (%) 11.7 (0e60) Macrophage (%) 47.9 (14.3e83.5) Lymphocytes (%) 3.1 (0e10.5) PTX3 (ng/mL) 1.2 (0e100) PTX3 above limit 14 (56) of detection
COPD (n ¼ 27) 10.6 58.8 2.3 35.3 1.4 24.6 24
Control (n ¼ 16)
P value
(1.5e32.1) 3.4 (0.4e6) <.001a, .004b (7.0e85.3) 23 (3e72) <.001a, .01b (0e18) 0.16 (0e1) .003c, .003b (11.7e87) 66 (22e94) .02c, <.001a (0e4) 3.6 (0.5e10.5) .012b, .01a (0e384) 0 (0e36) .009a, .012b (89) 3 (19) <.001a,b
Abbreviations: COPD, chronic obstructive pulmonary disease; PTX3, pentraxin 3; TCC, total cell count. a COPD vs controls. b Asthma vs COPD. c Asthma vs controls.
and eosinophil counts. Linear regression analyses were performed to examine the association between biomarkers and clinical variables and the decrease of %FEV1 (as the dependent variable) in patients with COPD. Variables significantly correlated with %FEV1 were imputed in linear regression analysis. Thus, 1 model was constructed that included sputum PTX3 levels, sputum neutrophil counts, age in years, and pack-years. Statistical significance was defined as a P value less than .05. Data were analyzed using SPSS 18.0 for Windows (SPSS, Inc, Chicago, Illinois). Results Patient Description As expected, patients with COPD were older and had significantly lower lung function compared with patients with asthma and healthy controls (Table 1). Smoking history was significantly more frequent in patients with COPD. COPD severity was classified as mild (stage I, n ¼ 1 [4%]), moderate (stage II, n ¼ 14 [52%]), severe (stage III, n ¼ 10 [37%]), or very severe (stage IV, n ¼ 2 [7%]) according to criteria of the Global Initiative for Chronic Obstructive Lung Disease.2 Sputum Biomarkers and COPD Diagnosis Compared with patients with asthma, healthy controls and patients with COPD had significantly increased total cell count and proportion of neutrophils (Table 2). As expected, subjects with asthma had larger proportions of eosinophils than patients with COPD and healthy control subjects (Table 2).
Figure 2. Induced sputum pentraxin 3 (iPTX3) levels in patients with chronic obstructive pulmonary disease (COPD) and patients with asthma with or without sputum neutrophilia (asthma with neutrophilia, n ¼ 7; COPD with neutrophilia, n ¼ 17).
Pentraxin 3 was detected in 88.9% of patients with COPD, 56.0% of patients with asthma, and 18.8% of controls (P ¼ .001; Table 2). It discriminated patients with COPD (24.6 ng/mL, 0e384 ng/mL) from controls (0 ng/mL, 0e36 ng/mL, P < .001) and from patients with asthma (1.2 ng/mL, 0e100 ng/mL, P ¼ .01; Fig 1), with an area under the receiver operating characteristics curve of 0.82 (0.71e0.94). PTX3 levels were significantly higher in participants with COPD and sputum neutrophilia (28.4 ng/mL, 11.3e304 ng/mL) compared with those without neutrophilia (11.9 ng/mL, 0e90.8 ng/mL, P ¼ .008; Fig 2). The discriminative value of sputum neutrophil levels was 0.81 (0.71e0.91). In patients with asthma and sputum neutrophilia, PTX3 levels were similar to those without neutrophilia (1.2 ng/mL [0e82 ng/mL] vs 0 ng/mL [0e14.3 ng/mL], respectively, P ¼ .6; Fig 2). PTX3 levels were not affected by the presence or absence of eosinophilia (data not shown). Regression analyses were performed to determine any independent association between biomarkers and COPD. Only sputum PTX3 and neutrophil counts were independently associated with COPD. Sputum Biomarkers and COPD Severity Because PTX3 was independently associated with COPD, the authors investigated whether some of these biomarkers were related to COPD severity (assessed by post-bronchodilator FEV1%). In a linear regression model, only pack-years (standardized b ¼ 0.436 [0.550 to 0.029], P ¼ .031) and PTX3 (standardized b ¼ 0.443 [5.78 to 0.37], P ¼ .028) were independently related to COPD severity. Discussion
Figure 1. Induced sputum pentraxin 3 (iPTX3) levels in patients with chronic obstructive pulmonary disease (COPD), patients with asthma, and healthy individuals.
In the present study, patients with COPD had significantly higher levels of PTX3 in induced sputum fluid phases than did those with asthma or healthy controls. In addition, PTX3 sputum levels were associated with sputum neutrophilia and inversely correlated with the magnitude of airflow obstruction only in patients with COPD. These findings suggest that PTX3 is a potential marker of airway inflammation in COPD. Despite great interest in the role of PTX3 in several inflammatory diseases, little is known about its role in respiratory diseases, including asthma and COPD.15e18 This understanding has been
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limited by conflicting results from the few clinical studies on this subject. Hamon et al15 found higher levels of PTX3 in the sputum of subjects with COPD compared with those with cystic fibrosis. In contrast, Van Pottelberge et al8 did not find any significant increase in PTX3 levels in the lungs of patients with COPD. An increase in PTX3 immunoreactivity has been observed in bronchial tissues of patients with allergic asthma compared with healthy controls.18 In these patients, PTX3 was localized mainly in the smooth muscle but was not related to the severity of the disease.18 In patients with COPD, but not in those with heart failure, plasma PTX3 levels were correlated with IL-1b levels.16 These dissimilarities between studies could reflect several aspects of PTX3 production, such as the presence of infection, site of production, measurement, and cells involved in its production. The present results support the role of PTX3 in sputum levels as a marker of COPD and its severity. Pentraxin 3 levels are almost undetectable in the blood and tissues of healthy individuals and increases rapidly in response to inflammatory stimuli in different pathologies, such as autoimmune diseases, septic shock,19 acute myocardial infarction,20e22 and traumatic brain injury.23 PTX3 knockout mice respond equally to lipopolysaccharide as wild-type controls.24 In contrast, mice that overexpress PTX3 are protected against sepsis induced by cecal ligation, perforation, and lipopolysaccharide.7 In the present study, all patients were free from a recent exacerbation and infection (in the 4 weeks before the study), but induced sputum fluid phase levels of PTX3 were significantly higher in the COPD group than in the asthma group. These results support a truly differential effect of COPD-related inflammation and it is not as a result of an enhanced PTX3 secretion in response to infection. However, these results cannot exclude that the PTX3 high levels seen in patients with COPD are related to local microbiomes and further studies are required to investigate this possibility. Nevertheless, one could presume that the presence of PTX3 indicates a greater degree of lung inflammation. In this context, PTX3 is highly specific to macrophages and neutrophils, and the present study showed that it is relatively specific for COPD-associated lung inflammation compared with patients with asthma and normal subjects. The issue of PTX3 being expressed in the lungs is not new and many experimental studies support this notion. For instance, lung epithelial cells stimulated by tumor necrosis factor-a (TNF-a) induced PTX3 production in human and mice.23,25 Also, PTX3 protein was found expressed constitutively by human airway smooth muscle cells and significantly upregulated by TNF-a and IL-1b, but not by TH2 (IL-4, IL-9, IL-13), TH1 (interferon-g), or TH17 (IL-17) cytokines.25 After exposure to TNF-a, nuclear factor-kB interacts with the human PTX3 promoter, at least in fibroblasts26 and epithelial cells.27 Furthermore, in lung epithelial cells stimulated by TNF-a, the inhibition of c-Jun N-terminal kinase pathway decreased PTX3 expression.27 The results of clinical studies are discrepant, which might be due to differences in study populations, disease severity, and methods. The present study has some limitations. First, the cross-sectional design is insufficient to infer causality (eg, PTX3 induces inflammation in patients with COPD). Second, the study design does not allow the determination of the temporal evolution of PTX3 levels and disease progression. Third, PTX3 levels in the sputum, and not in the lung, were investigated; thus, the authors cannot ascertain whether PTX3 is really produced by neutrophils, although it is very likely. Despite these limitations, this approach is clinically relevant and could add useful information to COPD diagnosis and probably to asthmaeCOPD overlap syndrome. Fourth, the authors cannot exclude the effect of treatment on PTX3 sputum levels because approximately half the patients were using inhaled corticosteroids or a long-acting b2-agonist. The control subjects and patients with
asthma were significantly younger than the patients with COPD. Some age-related diseases can increase PTX3 levels (eg, coronary artery disease), and aging is associated with increased inflammation. This fact might have led to a misinterpretation of the results. However, because PTX3 levels were measured in the sputum and PTX3 is produced by inflammatory cells (and not, for example, by the liver), it was expected that the sputum variation of PTX3 levels would be related mainly to airway inflammation and not to systemic inflammation or aging. In addition, only a weak correlation was observed between age and PTX3 in the present sample (r ¼ 0.36). Furthermore, the regression model did not alter the association between PTX3 and COPD (age was inclusive). In conclusion, the present study showed that PTX3 in sputum levels was significantly increased in patients with COPD and showed a good discriminatory power distinguishing these patients from patients with asthma and healthy individuals. These results open the perspective of using PTX3 in future studies to better characterize overlap syndrome.
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