Discussion
Although tobacco smoke is inhaled though the lung, there is substantial evidence that tobacco exposure has systemic manifestations and is associated with extra-pulmonary disease [
2‐
5]. While the mechanisms of tobacco smoke-induced systemic injury are not fully understood, inflammation is thought to play a key role. This study is one of largest multiplex investigations of cytokines and chemokine biomarkers to date and is one of the few that includes two large, independent, well phenotyped cohorts of current and former smokers. Although we found that most of the cytokines and chemokines were associated with some COPD phenotypes, only eotaxin and IL-6 were strongly and consistently associated with airflow flow limitation in both cohorts, even after adjustment for important clinical covariates. None of the nine biomarkers were associated with chronic bronchitis. None of the 9 biomarkers were consistently associated with COPD exacerbations, which is similar to what has been recently reported for COPD exacerbations in a more extensive study of other blood biomarkers, albeit with few subjects [
40].
Eotaxin-1 (CCL11) is a potent eosinophil chemoattractant that is normally associated with asthma [
41], but is known to play a role in other mucosal diseases such as inflammatory bowel disease (see review [
42]). Eotaxin and eotaxin receptor (CCR3) positive cells are higher in acute exacerbations of chronic bronchitis as well as asthma [
43]; however, the published associations between plasma eotaxin and COPD in non-exacerbating subjects are contradictory, possibly because most include only a small number of subjects. For instance, in 50 FORTE study participants (34 stable and 16 rapid decliners) and 11 controls, plasma eotaxin-1 was lower in rapid decliners compared to stable COPD patients, but eotaxin was also significantly lower in stable COPD subjects compared to normal controls (
p < 0.03) [
44]. In a different study of 21 COPD subjects and 9 controls, eotaxin was higher in COPD patients compared to controls [
45]. In our study, which included more than 3000 subjects, eotaxin was higher in COPD subjects in both cohorts compared to control subjects with no COPD and a comparable smoking history. Eotaxin was higher in subjects with chronic bronchitis and was positively associated with neutrophils and negatively associated with eosinophil counts. These findings suggest that eotaxin is associated with a neutrophilic/inflammatory COPD, but does not appear to be independently associated exacerbations or higher eosinophils, as might be expected with asthma exacerbations.
Another strong association was between IL-6 and COPD affection status, airflow limitation and emphysema progression. IL-6 is a 26 kDa, 184 amino acid multifunctional glycoprotein and pro-inflammatory cytokine that is produced in a variety of stromal and immune cells and which is associated with a large number of pulmonary and extra-pulmonary inflammatory diseases (see reviews [
46,
47]). In this study, which is appreciably larger than previously published studies, we found that IL-6 was associated with both case-control status, COPD severity, rate of decline in spirometry, and independently associated with emphysema progression as assessed by CT scans; however, it was not independently associated with exacerbations. The case-control associations are consistent with several large population studies. For example, in the Health, Aging, and Body Composition study which included 3075 subjects [
48], the Framingham Heart Study which included 2553 subjects [
49], the Rotterdam Study which included 572 older subjects [
50], plasma IL-6 was higher in those with COPD compared to those without. This is consistent with a recent meta-analysis of IL-6 and COPD, which included 1891 COPD subjects and 4946 controls from 33 studies [
51]. This meta-analysis also reported a non-statistically significant trend toward the mild-moderate COPD subjects having lower plasma IL-6 compared to severe COPD subjects; however, IL-6 was not associated with disease severity in 1793 subjects from in the ECLIPSE, which primarily included COPD subjects [
14]. IL-6 was also not associated with decline in the ECLIPSE cohort. Since IL-6 was strongly associated with neutrophils in both cohorts, this would suggest that IL-6 may drive the inflammatory phenotype which promotes progressive airflow limitation. While our analysis showed a statistically significant independent association with decline in lung function, adding IL-6 to the model with clinical covariates (e.g. low FEV
1%) added only about 4–5% to the explanation of variance. This is consistent with the concept that subjects with low lung function have an inflammatory phenotype and are predisposed to more rapid decline in lung function, and that adding biomarkers to these prediction models will add a small, but additional benefit to predicting decline on top of clinical covariates.
The COPDGene study is one of the largest current and former smoker cohorts with long term CT follow up and this study is one of the first to report IL-6 as an independent biomarker of emphysema progression. A pathologic role for IL-6 is supported by several observations. First, IL-6 binds to IL-6 receptor and signals through at gp130 subunit; it transduces inflammatory gene transcription through JAK-STAT pathways. Second, genetic blockade of the IL-6 receptor subunit gp130 blocks cigarette smoke induced emphysema [
52]. Third, IL-6 is associated with cardiovascular disease in COPD patients [
53] and recent literature supports a vascular etiology of emphysema [
54]. Although IL-6 specific treatments (e.g. tocilizumab) have been developed, but not yet tried as a treatment for COPD, one case report describes worsening of emphysema during treatment for rheumatoid arthritis [
55]. Thus, anti-IL-6 treatment in COPD should be done with caution.
In additional to eotaxin and IL-6, IL-2, IL-8, and IL-10 were also found to be elevated in COPD patients, although they accounted for only a small amount of the variance in airflow obstruction compared to IL-6 and eotaxin. For several of these cytokines, there are only smaller studies previously published. In a study of 10 COPD patients and 10 controls, ex vivo IL-2 release from stimulated T-cells was higher in COPD patients compared to smoking controls [
56]. In the 50 FORTE study participants discussed above, IL-2 was higher in COPD patients, but was lower in rapid decliners compared to stable COPD patients [
44]. Similarly, in small studies IL-8 has been reported to be elevated in COPD patients in smaller studies with less than 100 subjects [
57,
58]. This is the first large study to show that IL-8 is independently associated with progression of emphysema by CT scan and additional studies in independent longitudinal COPD cohorts should consider measuring IL-8. Similarly, we find that IL-10 is associated with worse COPD; however, there are only a few published studies, which may be underpowered to confirm or refute these observations. For example in a study of 94 COPD patients and 45 controls, IL-10 was no different between COPD patients and controls, but lower than in healthy non-smokers [
59]. Since IL-10 was not associated with progression of COPD or emphysema, it is unclear whether it may be a useful predictive marker.
Although CCL17 is more expressed in airway cells from COPD patients and plays a role in Th2 inflammation [
60], we found no association with any COPD phenotypes. However, our study is the first report of the rs9302690 SNP being a pQTL for CCL17, with the minor allele being associated with higher plasma levels of CCL17. This finding may be relevant to other clinical investigators because
CCL17 is expressed in many tissues and has been associated with atopic dermatitis [
61] and Hodgkin’s Lymphoma [
62]. In GTex analysis, the rs9302690 SNP is also a gene expression QTL (eQTL) (GTEx V6p) with the minor allele being associated with higher CCL mRNA in esophagus and testes and lower expression in adrenal and pituitary tissue. Thus, both CCL17 gene and protein expression should be adjusted for the rs9302690 genotype.
While this study was unique in that it featured two large well-characterized cohorts, confirmed strong associations of IL-6 and eotaxin, identified new pQTL SNPs, and identified potentially new biomarkers of COPD and emphysema progression, there were some important limitations. Most importantly, biomarkers were assessed at only a single time point and thus one cannot determine whether the biomarkers temporally fluctuate with disease activity. We also only studied 9 biologically plausible biomarkers, but there are new platforms which will permit the simultaneous measurements of hundreds or thousands of proteins, even if these platforms may not be designed to assay low abundant proteins such as interleukins. Also, although subgrouping into phenotypes showed that some cytokines such as IL-6 were associated with severity and progression of airflow obstruction and emphysema even in subjects without COPD or emphysema at baseline, other subgroup analyses were limited by the loss of power that occurred when subgroup sizes dropped below 500 subjects. This might suggest that biomarkers might be useful markers of disease progression in current and former smokers who do not yet manifest COPD or emphysema. Finally, other limitations of this study include the relatively low number of nonsmokers and only limited progression data in one of the cohorts (SPIROMICS).
Acknowledgements
COPDGene Acknowledgements
COPDGene® Investigators – Core Units
Administrative Center: James D. Crapo, MD (PI); Edwin K. Silverman, MD, PhD (PI); Barry J. Make, MD; Elizabeth A. Regan, MD, PhD.
Genetic Analysis Center: Terri Beaty, PhD; Ferdouse Begum, PhD; Robert Busch, MD; Peter J. Castaldi, MD, MSc; Michael Cho, MD; Dawn L. DeMeo, MD, MPH; Adel R. Boueiz, MD; Marilyn G. Foreman, MD, MS; Eitan Halper-Stromberg; Nadia N. Hansel, MD, MPH; Megan E. Hardin, MD; Lystra P. Hayden, MD, MMSc; Craig P. Hersh, MD, MPH; Jacqueline Hetmanski, MS, MPH; Brian D. Hobbs, MD; John E. Hokanson, MPH, PhD; Nan Laird, PhD; Christoph Lange, PhD; Sharon M. Lutz, PhD; Merry-Lynn McDonald, PhD; Margaret M. Parker, PhD; Dandi Qiao, PhD; Elizabeth A. Regan, MD, PhD; Stephanie Santorico, PhD; Edwin K. Silverman, MD, PhD; Emily S. Wan, MD; Sungho Won.
Imaging Center: Mustafa Al Qaisi, MD; Harvey O. Coxson, PhD; Teresa Gray; MeiLan K. Han, MD, MS; Eric A. Hoffman, PhD; Stephen Humphries, PhD; Francine L. Jacobson, MD, MPH; Philip F. Judy, PhD; Ella A. Kazerooni, MD; Alex Kluiber; David A. Lynch, MB; John D. Newell, Jr., MD; Elizabeth A. Regan, MD, PhD; James C. Ross, PhD; Raul San Jose Estepar, PhD; Joyce Schroeder, MD; Jered Sieren; Douglas Stinson; Berend C. Stoel, PhD; Juerg Tschirren, PhD; Edwin Van Beek, MD, PhD; Bram van Ginneken, PhD; Eva van Rikxoort, PhD; George Washko, MD; Carla G. Wilson, MS.
PFT QA Center, Salt Lake City, UT: Robert Jensen, PhD.
Data Coordinating Center and Biostatistics, National Jewish Health, Denver, CO: Douglas Everett, PhD; Jim Crooks, PhD; Camille Moore, PhD; Matt Strand, PhD; Carla G. Wilson, MS.
Epidemiology Core, University of Colorado Anschutz Medical Campus, Aurora, CO: John E. Hokanson, MPH, PhD; John Hughes, PhD; Gregory Kinney, MPH, PhD; Sharon M. Lutz, PhD; Katherine Pratte, MSPH; Kendra A. Young, PhD.
COPDGene® Investigators – Clinical Centers
Ann Arbor VA: Jeffrey L. Curtis, MD; Carlos H. Martinez, MD, MPH; Perry G. Pernicano, MD.
Baylor College of Medicine, Houston, TX: Nicola Hanania, MD, MS; Philip Alapat, MD; Mustafa Atik, MD; Venkata Bandi, MD; Aladin Boriek, PhD; Kalpatha Guntupalli, MD; Elizabeth Guy, MD; Arun Nachiappan, MD; Amit Parulekar, MD.
Brigham and Women’s Hospital, Boston, MA: Dawn L. DeMeo, MD, MPH; Craig Hersh, MD, MPH; Francine L. Jacobson, MD, MPH; George Washko, MD.
Columbia University, New York, NY: R. Graham Barr, MD, DrPH; John Austin, MD; Belinda D’Souza, MD; Gregory D.N. Pearson, MD; Anna Rozenshtein, MD, MPH, FACR; Byron Thomashow, MD.
Duke University Medical Center, Durham, NC: Neil MacIntyre, Jr., MD; H. Page McAdams, MD; Lacey Washington, MD.
HealthPartners Research Institute, Minneapolis, MN: Charlene McEvoy, MD, MPH; Joseph Tashjian, MD.
Johns Hopkins University, Baltimore, MD: Robert Wise, MD; Robert Brown, MD; Nadia N. Hansel, MD, MPH; Karen Horton, MD; Allison Lambert, MD, MHS; Nirupama Putcha, MD, MHS.
Los Angeles Biomedical Research Institute at Harbor UCLA Medical Center, Torrance, CA: Richard Casaburi, PhD, MD; Alessandra Adami, PhD; Matthew Budoff, MD; Hans Fischer, MD; Janos Porszasz, MD, PhD; Harry Rossiter, PhD; William Stringer, MD.
Michael E. DeBakey VAMC, Houston, TX: Amir Sharafkhaneh, MD, PhD; Charlie Lan, DO.
Minneapolis VA: Christine Wendt, MD; Brian Bell, MD.
Morehouse School of Medicine, Atlanta, GA: Marilyn G. Foreman, MD, MS; Eugene Berkowitz, MD, PhD; Gloria Westney, MD, MS.
National Jewish Health, Denver, CO: Russell Bowler, MD, PhD; David A. Lynch, MB.
Reliant Medical Group, Worcester, MA: Richard Rosiello, MD; David Pace, MD.
Temple University, Philadelphia, PA: Gerard Criner, MD; David Ciccolella, MD; Francis Cordova, MD; Chandra Dass, MD; Gilbert D’Alonzo, DO; Parag Desai, MD; Michael Jacobs, PharmD; Steven Kelsen, MD, PhD; Victor Kim, MD; A. James Mamary, MD; Nathaniel Marchetti, DO; Aditi Satti, MD; Kartik Shenoy, MD; Robert M. Steiner, MD; Alex Swift, MD; Irene Swift, MD; Maria Elena Vega-Sanchez, MD.
University of Alabama, Birmingham, AL: Mark Dransfield, MD; William Bailey, MD; Surya Bhatt, MD; Anand Iyer, MD; Hrudaya Nath, MD; J. Michael Wells, MD.
University of California, San Diego, CA: Joe Ramsdell, MD; Paul Friedman, MD; Xavier Soler, MD, PhD; Andrew Yen, MD.
University of Iowa, Iowa City, IA: Alejandro P. Comellas, MD; John Newell, Jr., MD; Brad Thompson, MD.
University of Michigan, Ann Arbor, MI: MeiLan K. Han, MD, MS; Ella Kazerooni, MD; Carlos H. Martinez, MD, MPH.
University of Minnesota, Minneapolis, MN: Joanne Billings, MD; Abbie Begnaud, MD; Tadashi Allen, MD.
University of Pittsburgh, Pittsburgh, PA: Frank Sciurba, MD; Jessica Bon, MD; Divay Chandra, MD, MSc; Carl Fuhrman, MD; Joel Weissfeld, MD, MPH.
University of Texas Health Science Center at San Antonio, San Antonio, TX: Antonio Anzueto, MD; Sandra Adams, MD; Diego Maselli-Caceres, MD; Mario E. Ruiz, MD.
SPIROMICS acknowledgements
The authors thank the SPIROMICS participants and participating physicians, investigators and staff for making this research possible. More information about the study and how to access SPIROMICS data is at
www.spiromics.org. We would like to acknowledge the following current and former investigators of the SPIROMICS sites and reading centers: Neil E Alexis, PhD; Wayne H Anderson, PhD; R Graham Barr, MD, DrPH; Eugene R Bleecker, MD; Richard C Boucher, MD; Russell P Bowler, MD, PhD; Elizabeth E Carretta, MPH; Stephanie A Christenson, MD; Alejandro P Comellas, MD; Christopher B Cooper, MD, PhD; David J Couper, PhD; Gerard J Criner, MD; Ronald G Crystal, MD; Jeffrey L Curtis, MD; Claire M Doerschuk, MD; Mark T Dransfield, MD; Christine M Freeman, PhD; MeiLan K Han, MD, MS; Nadia N Hansel, MD, MPH; Annette T Hastie, PhD; Eric A Hoffman, PhD; Robert J Kaner, MD; Richard E Kanner, MD; Eric C Kleerup, MD; Jerry A Krishnan, MD, PhD; Lisa M LaVange, PhD; Stephen C Lazarus, MD; Fernando J Martinez, MD, MS; Deborah A Meyers, PhD; John D Newell Jr., MD; Elizabeth C Oelsner, MD, MPH; Wanda K O’Neal, PhD; Robert Paine, III, MD; Nirupama Putcha, MD, MHS; Stephen I. Rennard, MD; Donald P Tashkin, MD; Mary Beth Scholand, MD; J Michael Wells, MD; Robert A Wise, MD; and Prescott G Woodruff, MD, MPH. The project officers from the Lung Division of the National Heart, Lung, and Blood Institute were Lisa Postow, PhD, and Thomas Croxton, PhD, MD.