Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)

Journal of Translational Medicine

Jeffrey L Curtis^1,11  Michelle Zeidler^1,10  Prescott G Woodruff⁴  Daichi Shimbo⁵  Stephen I Rennard¹  Robert Paine III⁶  Wanda K O’Neal²  Robert J Kaner³  Annette T Hastie⁷  David J Couper²  Elizabeth E Carretta²  Patricia V Basta²  Claire M Doerschuk^1,12  Neil E Alexis⁸  Joanne Sonstein^1,11  Jeanette P Brown^1,11  Valerie R Stolberg⁹  Sean Crudgington^1,11  Christine M Freeman^1,11 

[1] Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198, NE, USA;Marsico Lung Institute/University of North Carolina Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill 27599, NC, USA;Division of Pulmonary and Critical Care Medicine, Departments of Medicine and Genetic Medicine, Weill Cornell Medical College, New York 10021, NY, USA;Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of California at San Francisco, San Francisco 94143, CA, USA;Department of Medicine, Columbia University Medical Center, New York 10032, NY, USA;Division of Pulmonary, Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City 84112, UT, USA;Center for Genomics and Personalized Medicine, Wake Forest University, Winston-Salem 27157, NC, USA;Center for Environmental Medicine, Asthma, and Lung Biology, Chapel Hill 27599, NC, USA;Research Service, VA Ann Arbor Healthcare System, Ann Arbor, MI, USA;Division of Pulmonary, Critical Care, and Sleep Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles 90095, CA, USA;Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor 48109, MI, USA;Center for Airways Disease, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill 27599, NC, USA

关键词: Immunophenotyping;    Bronchoalveolar lavage;    Sputum;    Flow cytometry;    COPD;    Human;   

Others  :  1139413
DOI  :  10.1186/s12967-014-0374-z

 received in 2014-11-27, accepted in 2014-12-26,  发布年份 2015

【 摘 要 】

Background

Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument.

Methods

The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data.

Results

Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel.

Conclusions

Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters.

Trial registration

This study was registered with ClinicalTrials.gov as NCT01969344 webcite.

【 授权许可】

   
2015 Freeman et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Minino AM, Xu J, Kochanek KD: Deaths: preliminary data for 2008. Natl Vital Stat Rep 2010, 59:1-72.
• [2]Murray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990–2020: global burden of disease study. Lancet 1997, 349:1498-1504.
• [3]Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Pare PD: The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004, 350:2645-2653.
• [4]Barnes PJ, Celli BR: Systemic manifestations and comorbidities of COPD. Eur Respir J 2009, 33:1165-1185.
• [5]Han MK, Agusti A, Calverley PM, Celli BR, Criner G, Curtis JL, Fabbri LM, Goldin JG, Jones PW, Macnee W, et al.: Chronic obstructive pulmonary disease phenotypes: the future of COPD. Am J Respir Crit Care Med 2010, 182:598-604.
• [6]Couper D, Lavange LM, Han M, Barr RG, Bleecker E, Hoffman EA, Kanner R, Kleerup E, Martinez FJ, Woodruff PG, Rennard S: Design of the subpopulations and intermediate outcomes in COPD study (SPIROMICS). Thorax 2013, 69:491-494.
• [7]Giorgi JV, Cheng HL, Margolick JB, Bauer KD, Ferbas J, Waxdal M, Schmid I, Hultin LE, Jackson AL, Park L, et al.: The Multicenter AIDS Cohort Study Group: Quality control in the flow cytometric measurement of T-lymphocyte subsets: the multicenter AIDS cohort study experience. Clin Immunol Immunopathol 1990, 55:173-186.
• [8]Bradstock K, Matthews J, Benson E, Page F, Bishop J: Prognostic value of immunophenotyping in acute myeloid leukemia. Blood 1994, 84:1220-1225.
• [9]Benevolo G, Stacchini A, Spina M, Ferreri AJ, Arras M, Bellio L, Botto B, Bulian P, Cantonetti M, Depaoli L, et al.: Final results of a multicenter trial addressing role of CSF flow cytometric analysis in NHL patients at high risk for CNS dissemination. Blood 2012, 120:3222-3228.
• [10]Della Porta MG, Picone C, Pascutto C, Malcovati L, Tamura H, Handa H, Czader M, Freeman S, Vyas P, Porwit A, et al.: Multicenter validation of a reproducible flow cytometric score for the diagnosis of low-grade myelodysplastic syndromes: results of a European LeukemiaNET study. Haematologica 2012, 97:1209-1217.
• [11]Ng AA, Lee BT, Teo TS, Poidinger M, Connolly JE: Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials. J Immunol Methods 2012, 385:79-89.
• [12]Davis C, Wu X, Li W, Fan H, Reddy M: Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry. J Immunol Methods 2011, 363:158-165.
• [13]Alexis N, Soukup J, Ghio A, Becker S: Sputum phagocytes from healthy individuals are functional and activated: a flow cytometric comparison with cells in bronchoalveolar lavage and peripheral blood. Clin Immunol 2000, 97:21-32.
• [14]Majori M, Corradi M, Caminati A, Cacciani G, Bertacco S, Pesci A: Predominant Th1 cytokine pattern in peripheral blood from subjects with chronic obstructive pulmonary disease. J Allergy Clin Immunol 1999, 103:458-462.
• [15]Grumelli S, Corry DB, Song LZ, Song L, Green L, Huh J, Hacken J, Espada R, Bag R, Lewis DE, Kheradmand F: An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. PLoS Med 2004, 1:e8.
• [16]Sullivan AK, Simonian PL, Falta MT, Mitchell JD, Cosgrove GP, Brown KK, Kotzin BL, Voelkel NF, Fontenot AP: Oligoclonal CD4+ T cells in the lungs of patients with severe emphysema. Am J Respir Crit Care Med 2005, 172:590-596.
• [17]Freeman CM, Curtis JL, Chensue SW: CC chemokine receptor 5 and CXC chemokine receptor 6 expression by lung CD8+ cells correlates with chronic obstructive pulmonary disease severity. Am J Pathol 2007, 171:767-776.
• [18]Lee SH, Goswami S, Grudo A, Song LZ, Bandi V, Goodnight-White S, Green L, Hacken-Bitar J, Huh J, Bakaeen F, et al.: Antielastin autoimmunity in tobacco smoking-induced emphysema. Nat Med 2007, 13:567-569.
• [19]Freeman CM, Martinez FJ, Han MK, Washko GR Jr, McCubbrey AL, Chensue SW, Arenberg DA, Meldrum CA, McCloskey L, Curtis JL: Lung CD8+ T cells in COPD have increased expression of bacterial TLRs. Respir Res 2013, 14:13. BioMed Central Full Text
• [20]Lay JC, Peden DB, Alexis NE: Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways. Inhal Toxicol 2011, 23:392-406.
• [21]Leckie MJ, Jenkins GR, Khan J, Smith SJ, Walker C, Barnes PJ, Hansel TT: Sputum T lymphocytes in asthma, COPD and healthy subjects have the phenotype of activated intraepithelial T cells (CD69+ CD103+). Thorax 2003, 58:23-29.
• [22]Heron M, Grutters JC, ten Dam-Molenkamp KM, Hijdra D, van Heugten-Roeling A, Claessen AM, Ruven HJ, van den Bosch JM, van Velzen-Blad H: Bronchoalveolar lavage cell pattern from healthy human lung. Clin Exp Immunol 2012, 167:523-531.
• [23]Hiemstra PS: Altered macrophage function in chronic obstructive pulmonary disease. Ann Am Thorac Soc 2013, 10(Suppl):S180-S185.
• [24]Woodruff PG, Koth LL, Yang YH, Rodriguez MW, Favoreto S, Dolganov GM, Paquet AC, Erle DJ: A distinctive alveolar macrophage activation state induced by cigarette smoking. Am J Respir Crit Care Med 2005, 172:1383-1392.
• [25]Shaykhiev R, Krause A, Salit J, Strulovici-Barel Y, Harvey BG, O'Connor TP, Crystal RG: Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease. J Immunol 2009, 183:2867-2883.
• [26]Gautier EL, Shay T, Miller J, Greter M, Jakubzick C, Ivanov S, Helft J, Chow A, Elpek KG, Gordonov S, et al.: Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol 2012, 13:1118-1128.
• [27]Guth AM, Janssen WJ, Bosio CM, Crouch EC, Henson PM, Dow SW: Lung environment determines unique phenotype of alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 2009, 296:L936-L946.
• [28]Ziegler-Heitbrock L: The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007, 81:584-592.
• [29]Chow A, Brown BD, Merad M: Studying the mononuclear phagocyte system in the molecular age. Nat Rev Immunol 2011, 11:788-798.
• [30]Moniuszko M, Bodzenta-Lukaszyk A, Kowal K, Lenczewska D, Dabrowska M: Enhanced frequencies of CD14++CD16+, but not CD14 + CD16+, peripheral blood monocytes in severe asthmatic patients. Clin Immunol 2009, 130:338-346.
• [31]Ziegler-Heitbrock L, Hofer TP: Toward a refined definition of monocyte subsets. Front Immunol 2013, 4:23.
• [32]Hijdra D, Vorselaars AD, Grutters JC, Claessen AM, Rijkers GT: Phenotypic characterization of human intermediate monocytes. Front Immunol 2013, 4:339.
• [33]Brittan M, Barr L, Conway Morris A, Duffin R, Rossi F, Johnston S, Monro G, Anderson N, Rossi AG, McAuley DF, et al.: A novel subpopulation of monocyte-like cells in the human lung after lipopolysaccharide inhalation. Eur Respir J 2012, 40:206-214.
• [34]Demedts IK, Brusselle GG, Vermaelen KY, Pauwels RA: Identification and characterization of human pulmonary dendritic cells. Am J Respir Cell Mol Biol 2005, 32:177-184.
• [35]Freeman CM, Martinez FJ, Han MK, Ames TM, Chensue SW, Todt JC, Arenberg DA, Meldrum CA, Getty C, McCloskey L, Curtis JL: Lung dendritic cell expression of maturation molecules increases with worsening chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2009, 180:1179-1188.
• [36]Jonigk D, Al-Omari M, Maegel L, Muller M, Izykowski N, Hong J, Hong K, Kim SH, Dorsch M, Mahadeva R, et al.: Anti-inflammatory and immunomodulatory properties of alpha1-antitrypsin without inhibition of elastase. Proc Natl Acad Sci U S A 2013, 110:15007-15012.
• [37]Wright AK, Rao S, Range S, Eder C, Hofer TP, Frankenberger M, Kobzik L, Brightling C, Grigg J, Ziegler-Heitbrock L: Pivotal advance: expansion of small sputum macrophages in CF: failure to express MARCO and mannose receptors. J Leukoc Biol 2009, 86:479-489.
• [38]Orr Y, Taylor JM, Bannon PG, Geczy C, Kritharides L: Circulating CD10-/CD16low neutrophils provide a quantitative index of active bone marrow neutrophil release. Br J Haematol 2005, 131:508-519.
• [39]Brightling CE, McKenna S, Hargadon B, Birring S, Green R, Siva R, Berry M, Parker D, Monteiro W, Pavord ID, Bradding P: Sputum eosinophilia and the short term response to inhaled mometasone in chronic obstructive pulmonary disease. Thorax 2005, 60:193-198.
• [40]Brightling CE, Monteiro W, Ward R, Parker D, Morgan MD, Wardlaw AJ, Pavord ID: Sputum eosinophilia and short-term response to prednisolone in chronic obstructive pulmonary disease: a randomised controlled trial. Lancet 2000, 356:1480-1485.
• [41]Siva R, Green RH, Brightling CE, Shelley M, Hargadon B, McKenna S, Monteiro W, Berry M, Parker D, Wardlaw AJ, Pavord ID: Eosinophilic airway inflammation and exacerbations of COPD: a randomised controlled trial. Eur Respir J 2007, 29:906-913.
• [42]Stein ML, Villanueva JM, Buckmeier BK, Yamada Y, Filipovich AH, Assa'ad AH, Rothenberg ME: Anti-IL-5 (mepolizumab) therapy reduces eosinophil activation ex vivo and increases IL-5 and IL-5 receptor levels. J Allergy Clin Immunol 2008, 121:1473-1483. 1483 e1471-1474
• [43]Davoine F, Labonte I, Ferland C, Mazer B, Chakir J, Laviolette M: Role and modulation of CD16 expression on eosinophils by cytokines and immune complexes. Int Arch Allergy Immunol 2004, 134:165-172.
• [44]Atar OD, Eisert C, Pokov I, Serebruany VL: Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry. J Thromb Thrombolysis 2010, 30:79-83.
• [45]Stewart JC, Villasmil ML, Frampton MW: Changes in fluorescence intensity of selected leukocyte surface markers following fixation. Cytometry A 2007, 71:379-385.
• [46]Hodge S, Hodge G, Nairn J, Holmes M, Reynolds PN: Increased airway granzyme B and perforin in current and ex-smoking COPD subjects. COPD 2006, 3:179-187.
• [47]Kim WD, Chi HS, Choe KH, Oh YM, Lee SD, Kim KR, Yoo KH, Ngan DA, Elliott WM, Granville DJ, et al.: A possible role for CD8+ and non-CD8+ cell granzyme B in early small airway wall remodelling in centrilobular emphysema. Respirology 2013, 18:688-696.