Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway
B R Leaker, G C Nicholson, F Y Ali, N Daudi, B J O'Connor, P J Barnes, B R Leaker, G C Nicholson, F Y Ali, N Daudi, B J O'Connor, P J Barnes
Abstract
Background: Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.
Methods: Eight healthy smokers aged 40-65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.
Results: A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.
Conclusions: The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.
Trial registration: NHS Health Research Authority (13/LO/0256).
Figures
![Fig. 1](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/4559920/bin/12931_2015_268_Fig1_HTML.jpg)
![Fig. 2](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/4559920/bin/12931_2015_268_Fig2_HTML.jpg)
![Fig. 3](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/4559920/bin/12931_2015_268_Fig3_HTML.jpg)
![Fig. 4](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/4559920/bin/12931_2015_268_Fig4_HTML.jpg)
References
- Barnes PJ, Chowdhury B, Kharitonov SA, Magnussen H, Page CP, Postma D, Saetta M. Pulmonary biomarkers in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2006;174:6–14. doi: 10.1164/rccm.200510-1659PP.
- Kostikas K, Bakakos P, Papiris S, Stolz D, Celli BR. Systemic biomarkers in the evaluation and management of COPD patients: are we getting closer to clinical application? Curr Drug Targets. 2013;14:177–191. doi: 10.2174/1389450111314020005.
- Baines KJ, Pavord ID, Gibson PG. The role of biomarkers in the management of airways disease. Int J Tuberc Lung Dis. 2014;18:1264–1268. doi: 10.5588/ijtld.14.0226.
- Cazzola M, MacNee W, Martinez FJ, Rabe KF, Franciosi LG, Barnes PJ, Brusasco V, Burge PS, Calverley PM, Celli BR, et al. Outcomes for COPD pharmacological trials: from lung function to biomarkers. Eur Respir J. 2008;31:416–469. doi: 10.1183/09031936.00099306.
- Maestrelli P, Saetta M, Di Stefano A, Calcagni PG, Turato G, Ruggieri MP, Roggeri A, Mapp CE, Fabbri LM. Comparison of leukocyte counts in sputum, bronchial biopsies, and bronchoalveolar lavage. Am J Respir Crit Care Med. 1995;152:1926–1931. doi: 10.1164/ajrccm.152.6.8520757.
- Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006;7:3. doi: 10.1186/1471-2199-7-3.
- Barnes PJ. New concepts in chronic obstructive pulmonary disease. Annu Rev Med. 2003;54:113–129. doi: 10.1146/annurev.med.54.101601.152209.
- Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol. 2008;8:183–192. doi: 10.1038/nri2254.
- Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Paré PD. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004;350:2645–2653. doi: 10.1056/NEJMoa032158.
- Franciosi LG, Page CP, Celli BR, Cazzola M, Walker MJ, Danhof M, Rabe KF, Della Pasqua OE. Markers of disease severity in chronic obstructive pulmonary disease. Pulm Pharmacol Ther. 2006;19:189–199. doi: 10.1016/j.pupt.2005.05.001.
- Gelfand EW. Inflammatory mediators in allergic rhinitis. J Allergy Clin Immunol. 2004;114:S135–S138. doi: 10.1016/j.jaci.2004.08.043.
- Meltzer EO, Szwarcberg J, Pill MW. Allergic rhinitis, asthma, and rhinosinusitis: diseases of the integrated airway. J Manag Care Pharm. 2004;10:310–317.
- Compalati E, Ridolo E, Passalacqua G, Braido F, Villa E, Canonica GW. The link between allergic rhinitis and asthma: the united airways disease. Expert Rev Clin Immunol. 2010;6:413–423. doi: 10.1586/eci.10.15.
- Braunstahl GJ. The unified immune system: respiratory tract-nasobronchial interaction mechanisms in allergic airway disease. J Allergy Clin Immunol. 2005;115:142–148. doi: 10.1016/j.jaci.2004.10.041.
- Serrano C, Valero A, Picado C. Rhinitis and asthma: one airway, one disease. Arch Bronconeumol. 2005;41:569–578. doi: 10.1157/13079841.
- Wagener AH, Zwinderman AH, Luiten S, Fokkens WJ, Bel EH, Sterk PJ, van Drunen CM. The impact of allergic rhinitis and asthma on human nasal and bronchial epithelial gene expression. PLoS One. 2013;8:e80257. doi: 10.1371/journal.pone.0080257.
- Haslam PL, Baughman RP. Report of ERS Task Force: guidelines for measurement of acellular components and standardization of BAL. Eur Respir J. 1999;14:245–248. doi: 10.1034/j.1399-3003.1999.14b01.x.
- Meyer KC, Raghu G, Baughman RP, Brown KK, Costabel U, du Bois RM, Drent M, Haslam PL, Kim DS, Nagai S, et al. An official American Thoracic Society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease. Am J Respir Crit Care Med. 2012;185:1004–1014. doi: 10.1164/rccm.201202-0320ST.
- Faul JL, Demers EA, Burke CM, Poulter LW. The reproducibility of repeat measures of airway inflammation in stable atopic asthma. Am J Respir Crit Care Med. 1999;160:1457–1461. doi: 10.1164/ajrccm.160.5.9812027.
- Olsen HH, Grunewald J, Tornling G, Skold CM, Eklund A. Bronchoalveolar lavage results are independent of season, age, gender and collection site. PLoS One. 2012;7:e43644. doi: 10.1371/journal.pone.0043644.
- Foster MW, Thompson JW, Que LG, Yang IV, Schwartz DA, Moseley MA, Marshall HE. Proteomic analysis of human bronchoalveolar lavage fluid after subsgemental exposure. J Proteome Res. 2013;12:2194–2205. doi: 10.1021/pr400066g.
- Baughman RP. The uncertainties of bronchoalveolar lavage. Eur Respir J. 1997;10:1940–1942. doi: 10.1183/09031936.97.10091940.
- Baughman RP. Technical aspects of bronchoalveolar lavage: recommendations for a standard procedure. Semin Respir Crit Care Med. 2007;28:475–485. doi: 10.1055/s-2007-991520.
- Lofdahl JM, Cederlund K, Nathell L, Eklund A, Skold CM. Bronchoalveolar lavage in COPD: fluid recovery correlates with the degree of emphysema. Eur Respir J. 2005;25:275–281. doi: 10.1183/09031936.05.00033504.
- Naclerio RM, Baroody FM. Response of nasal mucosa to histamine or methacholine challenge: use of a quantitative method to examine the modulatory effects of atropine and ipratropium bromide. J Allergy Clin Immunol. 1992;90:1051–1054. doi: 10.1016/0091-6749(92)90122-I.
- Sim TC, Reece LM, Hilsmeier KA, Grant JA, Alam R. Secretion of chemokines and other cytokines in allergen-induced nasal responses: inhibition by topical steroid treatment. Am J Respir Crit Care Med. 1995;152:927–933. doi: 10.1164/ajrccm.152.3.7545059.
- Alam R, Sim TC, Hilsmeier K, Grant JA. Development of a new technique for recovery of cytokines from inflammatory sites in situ. J Immunol Methods. 1992;155:25–29. doi: 10.1016/0022-1759(92)90267-W.
- Leonidas NC, Marcella R, Brianr RL, Nicole AC, Robin M, Grant CN, et al. Effects Of Single Doses Of Oral Corticosteroid On Biomarkers Of Nasal Allergen Challenge (NAC). In A36 INFLAMMATION AND THE AIRWAY EPITHELIUM. American Thoracic Society: A1402-A: American Thoracic Society International Conference Abstracts].
- Todd JL, Goldstein DB, Ge D, Christie J, Palmer SM. The state of genome-wide association studies in pulmonary disease: a new perspective. Am J Respir Crit Care Med. 2011;184:873–880. doi: 10.1164/rccm.201106-0971PP.
- COPD Genetics | COPDGene []. Accessed 02 Sept 2015.
- Qiu W, Cho MH, Riley JH, Anderson WH, Singh D, Bakke P, Gulsvik A, Litonjua AA, Lomas DA, Crapo JD, et al. Genetics of sputum gene expression in chronic obstructive pulmonary disease. PLoS One. 2011;6:e24395. doi: 10.1371/journal.pone.0024395.
- Fabbri LM, Luppi F, Beghe B, Rabe KF. Complex chronic comorbidities of COPD. Eur Respir J. 2008;31:204–212. doi: 10.1183/09031936.00114307.
- Toraldo DM, Nuccio FD, Scoditti E. Systemic Inflammation in Chronic Obstructive Pulmonary Disease: May Diet Play a Therapeutic Role? J Allergy Ther. 2013;S2:005.
- Caramori G, Adcock IM, Di Stefano A, Chung KF. Cytokine inhibition in the treatment of COPD. Int J Chron Obstruct Pulmon Dis. 2014;9:397–412.
- Traves SL, Culpitt SV, Russell RE, Barnes PJ, Donnelly LE. Increased levels of the chemokines GROalpha and MCP-1 in sputum samples from patients with COPD. Thorax. 2002;57:590–595. doi: 10.1136/thorax.57.7.590.
- Capelli A, Di Stefano A, Gnemmi I, Balbo P, Cerutti CG, Balbi B, Lusuardi M, Donner CF. Increased MCP-1 and MIP-1beta in bronchoalveolar lavage fluid of chronic bronchitics. Eur Respir J. 1999;14:160–165. doi: 10.1034/j.1399-3003.1999.14a27.x.
- Tanino M, Betsuyaku T, Takeyabu K, Tanino Y, Yamaguchi E, Miyamoto K, Nishimura M. Increased levels of interleukin-8 in BAL fluid from smokers susceptible to pulmonary emphysema. Thorax. 2002;57:405–411. doi: 10.1136/thorax.57.5.405.
- Smyth LJ, Starkey C, Gordon FS, Vestbo J, Singh D. CD8 chemokine receptors in chronic obstructive pulmonary disease. Clin Exp Immunol. 2008;154:56–63. doi: 10.1111/j.1365-2249.2008.03729.x.
- Brozyna S, Ahern J, Hodge G, Nairn J, Holmes M, Reynolds PN, Hodge S. Chemotactic mediators of Th1 T-cell trafficking in smokers and COPD patients. Copd. 2009;6:4–16. doi: 10.1080/15412550902724164.
- Ying S, O’Connor B, Ratoff J, Meng Q, Fang C, Cousins D, Zhang G, Gu S, Gao Z, Shamji B, et al. Expression and cellular provenance of thymic stromal lymphopoietin and chemokines in patients with severe asthma and chronic obstructive pulmonary disease. J Immunol. 2008;181:2790–2798. doi: 10.4049/jimmunol.181.4.2790.
- Wesselius LJ, Nelson ME, Bailey K, O’Brien-Ladner AR. Rapid lung cytokine accumulation and neutrophil recruitment after lipopolysaccharide inhalation by cigarette smokers and nonsmokers. J Lab Clin Med. 1997;129:106–114. doi: 10.1016/S0022-2143(97)90167-0.
- Soler N, Ewig S, Torres A, Filella X, Gonzalez J, Zaubet A. Airway inflammation and bronchial microbial patterns in patients with stable chronic obstructive pulmonary disease. Eur Respir J. 1999;14:1015–1022. doi: 10.1183/09031936.99.14510159.
- Koyama S, Sato E, Haniuda M, Numanami H, Nagai S, Izumi T. Decreased level of vascular endothelial growth factor in bronchoalveolar lavage fluid of normal smokers and patients with pulmonary fibrosis. Am J Respir Crit Care Med. 2002;166:382–385. doi: 10.1164/rccm.2103112.
- Betsuyaku T, Nishimura M, Takeyabu K, Tanino M, Venge P, Xu S, Kawakami Y. Neutrophil granule proteins in bronchoalveolar lavage fluid from subjects with subclinical emphysema. Am J Respir Crit Care Med. 1999;159:1985–1991. doi: 10.1164/ajrccm.159.6.9809043.
- Glader P, Eldh B, Bozinovski S, Andelid K, Sjostrand M, Malmhall C, Anderson GP, Riise GC, Qvarfordt I, Linden A. Impact of acute exposure to tobacco smoke on gelatinases in the bronchoalveolar space. Eur Respir J. 2008;32:644–650. doi: 10.1183/09031936.00121507.
- Pesci A, Balbi B, Majori M, Cacciani G, Bertacco S, Alciato P, Donner CF. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur Respir J. 1998;12:380–386. doi: 10.1183/09031936.98.12020380.
- Kaur M, Smyth LJ, Cadden P, Grundy S, Ray D, Plumb J, Singh D. T lymphocyte insensitivity to corticosteroids in chronic obstructive pulmonary disease. Respir Res. 2012;13:20. doi: 10.1186/1465-9921-13-20.
- Hollander C, Sitkauskiene B, Sakalauskas R, Westin U, Janciauskiene SM. Serum and bronchial lavage fluid concentrations of IL-8, SLPI, sCD14 and sICAM-1 in patients with COPD and asthma. Respir Med. 2007;101:1947–1953. doi: 10.1016/j.rmed.2007.04.010.
- Cheng SL, Wang HC, Yu CJ, Yang PC. Increased expression of placenta growth factor in COPD. Thorax. 2008;63:500–506. doi: 10.1136/thx.2007.087155.
- Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ. Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2002;26:602–609. doi: 10.1165/ajrcmb.26.5.4685.
- Vlahos R, Wark PA, Anderson GP, Bozinovski S. Glucocorticosteroids differentially regulate MMP-9 and neutrophil elastase in COPD. PLoS One. 2012;7:e33277. doi: 10.1371/journal.pone.0033277.
- Barbieri SS, Zacchi E, Amadio P, Gianellini S, Mussoni L, Weksler BB, Tremoli E. Cytokines present in smokers’ serum interact with smoke components to enhance endothelial dysfunction. Cardiovasc Res. 2011;90:475–483. doi: 10.1093/cvr/cvr032.
- Song XY, Zhou SJ, Xiao N, Li YS, Zhen DZ, Su CY, Liu ZD. Research on the relationship between serum levels of inflammatory cytokines and non-small cell lung cancer. Asian Pac J Cancer Prev. 2013;14:4765–4768. doi: 10.7314/APJCP.2013.14.8.4765.
- de Torres JP, Casanova C, Pinto-Plata V, Varo N, Restituto P, Cordoba-Lanus E, Baz-Davila R, Aguirre-Jaime A, Celli BR. Gender differences in plasma biomarker levels in a cohort of COPD patients: a pilot study. PLoS One. 2011;6:e16021. doi: 10.1371/journal.pone.0016021.
- Aquilante CL, Beitelshees AL, Zineh I. Correlates of serum matrix metalloproteinase-8 (MMP-8) concentrations in nondiabetic subjects without cardiovascular disease. Clin Chim Acta. 2007;379:48–52. doi: 10.1016/j.cca.2006.12.006.
- Snitker S, Xie K, Ryan KA, Yu D, Shuldiner AR, Mitchell BD, Gong DW. Correlation of circulating MMP-9 with white blood cell count in humans: effect of smoking. PLoS One. 2013;8:e66277. doi: 10.1371/journal.pone.0066277.
- Pinto-Plata V, Casanova C, Mullerova H, de Torres JP, Corado H, Varo N, Cordoba E, Zeineldine S, Paz H, Baz R, et al. Inflammatory and repair serum biomarker pattern: association to clinical outcomes in COPD. Respir Res. 2012;13:71. doi: 10.1186/1465-9921-13-71.
- Tsai JJ, Liao EC, Hsu JY, Lee WJ, Lai YK. The differences of eosinophil- and neutrophil-related inflammation in elderly allergic and non-allergic chronic obstructive pulmonary disease. J Asthma. 2010;47:1040–1044.
- Boutou AK, Pitsiou GG, Stanopoulos I, Kontakiotis T, Kyriazis G, Argyropoulou P. Levels of inflammatory mediators in chronic obstructive pulmonary disease patients with anemia of chronic disease: a case–control study. QJM. 2012;105:657–663. doi: 10.1093/qjmed/hcs024.
- Valipour A, Schreder M, Wolzt M, Saliba S, Kapiotis S, Eickhoff P, Burghuber OC. Circulating vascular endothelial growth factor and systemic inflammatory markers in patients with stable and exacerbated chronic obstructive pulmonary disease. Clin Sci (Lond) 2008;115:225–232. doi: 10.1042/CS20070382.
Source: PubMed