Fibrocyte measurement in peripheral blood correlates with number of cultured mature fibrocytes in vitro and is a potential biomarker for interstitial lung disease in Rheumatoid Arthritis
Søren Andreas Just, Hanne Lindegaard, Eva Kildall Hejbøl, Jesper Rømhild Davidsen, Niels Bjerring, Søren Werner Karlskov Hansen, Henrik Daa Schrøder, Inger Marie Jensen Hansen, Torben Barington, Christian Nielsen, Søren Andreas Just, Hanne Lindegaard, Eva Kildall Hejbøl, Jesper Rømhild Davidsen, Niels Bjerring, Søren Werner Karlskov Hansen, Henrik Daa Schrøder, Inger Marie Jensen Hansen, Torben Barington, Christian Nielsen
Abstract
Background: Interstitial lung disease (ILD) can be a severe extra-articular disease manifestation in Rheumatoid Arthritis (RA). A potential role of fibrocytes in RA associated ILD (RA-ILD) has not previously been described. We present a modified faster method for measuring circulating fibrocytes, without intracellular staining. The results are compared to the traditional culture method, where the number of monocytes that differentiate into mature fibrocytes in vitro are counted. The results are following compared to disease activity in patients with severe asthma, ILD, RA (without diagnosed ILD) and RA with verified ILD (RA-ILD).
Method: CD45+ CD34+ CD11b+ (7-AAD- CD3- CD19- CD294-) cells were isolated by cell sorting and stained for pro-collagen type 1. Thirty-nine patients (10 RA, 9 ILD and 10 with severe asthma, 10 with RA-ILD) and 10 healthy controls (HC) were included. Current medication, disease activity, pulmonary function test and radiographic data were collected. Circulating fibrocytes were quantified by flow cytometry. Peripheral blood mononuclear cells were isolated and cultured for 5 days and the numbers of mature fibrocytes were counted.
Results: 90.2% (mean, SD = 1.5%) of the sorted cells were pro-collagen type 1 positive and thereby fulfilled the criteria for being circulating fibrocytes. The ILD and RA-ILD groups had increased levels of circulating fibrocytes compared to HC (p < 0.05). Levels of circulating fibrocytes correlated overall to number of monocytes that subsequently in vitro differentiated to mature fibrocytes (r = 0.81, p < 0.001). RA patients with pathologically reduced diffusion capacity for carbon monoxide adjusted for hemoglobin (DLCOc) in both the RA and in the combined RA + RA-ILD group, had significantly higher levels of both circulating and number of cultured mature fibrocytes (both p < 0.05). In both groups, the level of circulating fibrocytes and number of mature fibrocytes in culture also correlated to a reduction in DLCOc (r = -0.61 an r = -0.58 both p < 0.05).
Conclusions: We presented a fast and valid method for measuring circulating fibrocytes using flow cytometry on lysed peripheral blood. Further, we showed for the first time, that the level of circulating fibrocytes correlated with the number of peripheral blood mononuclear cells, that differentiated into mature fibrocytes in vitro. Reduced DLCOc was correlated with high levels of circulating and mature fibrocytes in RA, which have not been reported previously. In such, this study suggests that fibrocytes may exhibit an important role in the pathogenesis of RA-ILD, which requires further clarification in future studies.
Trial registration: ClinicalTrials.gov : NCT02711657 , registered 13/3-2016, retrospectively registered.
Keywords: Fibrocytes; Interstitial lung disease; Rheumatoid arthritis.
Conflict of interest statement
Ethics approval and consent to participateThe study was approved by the Regional Committees on Health Research Ethics for Southern Denmark (ID: S-20150172) and the Danish Data Protection Agency (ID: 2008–58-0035) and registered on All participants gave written informed consent. Further, the study was a part of Odense University Hospital Patient data Explorative Network (OPEN) in which all data and forms are stored in accordance with OUH guidelines and the Data Protection Agency.
Consent for publicationNot applicable.
Competing interestsThe authors declare that they have no competing interests.
Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References
- Mathai SC, Danoff SK. Management of interstitial lung disease associated with connective tissue disease. BMJ. 2016;352:h6819. doi: 10.1136/bmj.h6819.
- Mehrad B, Burdick MD, Strieter RM. Fibrocyte CXCR4 regulation as a therapeutic target in pulmonary fibrosis. Int J Biochem Cell Biol. 2009;41:1708–1718. doi: 10.1016/j.biocel.2009.02.020.
- Daccord C, Maher TM. Recent advances in understanding idiopathic pulmonary fibrosis. F1000Res. 2016;5
- Galligan CL, Fish EN. The role of circulating fibrocytes in inflammation and autoimmunity. J Leukoc Biol. 2013;93:45–50. doi: 10.1189/jlb.0712365.
- Herzog EL, Bucala R. Fibrocytes in health and disease. Exp Hematol. 2010;38:548–556. doi: 10.1016/j.exphem.2010.03.004.
- Pilling D, Vakil V, Cox N, Gomer RH. TNF-alpha-stimulated fibroblasts secrete lumican to promote fibrocyte differentiation. Proc Natl Acad Sci U S A. 2015;112:11929–11934. doi: 10.1073/pnas.1507387112.
- White MJ, Galvis-Carvajal E, Gomer RH. A brief exposure to tryptase or thrombin potentiates fibrocyte differentiation in the presence of serum or serum amyloid p. J Immunol. 2015;194:142–150. doi: 10.4049/jimmunol.1401777.
- Peng H, Herzog EL. Fibrocytes: emerging effector cells in chronic inflammation. Curr Opin Pharmacol. 2012;12:491–496. doi: 10.1016/j.coph.2012.03.002.
- Guiot J, Moermans C, Henket M, Corhay JL, Louis R. Blood Biomarkers in Idiopathic Pulmonary Fibrosis. Lung. 2017;195(3):273-80.
- Moeller A, Gilpin SE, Ask K, Cox G, Cook D, Gauldie J, Margetts PJ, Farkas L, Dobranowski J, Boylan C, et al. Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2009;179:588–594. doi: 10.1164/rccm.200810-1534OC.
- Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 2004;114:438–446. doi: 10.1172/JCI200420997.
- Reich B, Schmidbauer K, Rodriguez Gomez M, Johannes Hermann F, Gobel N, Bruhl H, Ketelsen I, Talke Y, Mack M. Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model. Kidney Int. 2013;84:78–89. doi: 10.1038/ki.2013.84.
- Bianchetti L, Isgrò M, Marini MA, Bellini A, Schmidt M, Mattoli S. Enumeration of circulating fibrocytes for clinical use in asthma by an optimized single-platform flow cytometry assay. BBA Clin. 2014;1:52–58. doi: 10.1016/j.bbacli.2014.06.002.
- Shipe R, Burdick MD, Strieter BA, Liu L, Shim YM, Sung SS, Teague WG, Mehrad B, Strieter RM, Rose CE., Jr Number, activation, and differentiation of circulating fibrocytes correlate with asthma severity. J Allergy Clin Immunol. 2016;137:750–757. doi: 10.1016/j.jaci.2015.07.037.
- Mattoli S. Involvement of fibrocytes in asthma and clinical implications. Clin Exp Allergy. 2015;45(10):1497–1509. doi: 10.1111/cea.12525.
- Pilling D, Gomer RH. Differentiation of circulating monocytes into fibroblast-like cells. Methods Mol Biol. 2012;904:191–206.
- Cox N, Pilling D, Gomer RH. NaCl potentiates human fibrocyte differentiation. PLoS One. 2012;7:e45674. doi: 10.1371/journal.pone.0045674.
- Zhang H, Maric I, DiPrima MJ, Khan J, Orentas RJ, Kaplan RN, Mackall CL. Fibrocytes represent a novel MDSC subset circulating in patients with metastatic cancer. Blood. 2013;122:1105–1113. doi: 10.1182/blood-2012-08-449413.
- Raza K, Falciani F, Curnow SJ, Ross EJ, Lee CY, Akbar AN, Lord JM, Gordon C, Buckley CD, Salmon M. Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin. Arthritis Res Ther. 2005;7:R784–R795. doi: 10.1186/ar1733.
- Moore BB, Fry C, Zhou Y, Murray S, Han MK, Martinez FJ, Flaherty KR, The CI Inflammatory leukocyte phenotypes correlate with disease progression in idiopathic pulmonary fibrosis. Front Med. 2014;1:56. doi: 10.3389/fmed.2014.00056.
- McGee HS, Agrawal DK. TH2 cells in the pathogenesis of airway remodeling: regulatory T cells a plausible panacea for asthma. Immunol Res. 2006;35:219–232. doi: 10.1385/IR:35:3:219.
- Hu X, DeBiasi EM, Herzog EL. Flow Cytometric identification of Fibrocytes in the human circulation. Methods Mol Biol. 2015;1343:19–33. doi: 10.1007/978-1-4939-2963-4_3.
- Zhang H, Mackall C. Identifying an IDO-expressing “fibrocyte” subset of myeloid suppressor cells in pediatric cancer patients. J Immunol. 2012;188(1 Supplement):127.28.
- Maharaj S, Shimbori C, Kolb M. Fibrocytes in pulmonary fibrosis: a brief synopsis. Eur Respir Rev. 2013;22:552–557. doi: 10.1183/09059180.00007713.
- Fujiwara A, Kobayashi H, Masuya M, Maruyama M, Nakamura S, Ibata H, Fujimoto H, Ohnishi M, Urawa M, Naito M, et al. Correlation between circulating fibrocytes, and activity and progression of interstitial lung diseases. Respirology. 2012;17:693–698. doi: 10.1111/j.1440-1843.2012.02167.x.
- Galligan CL, Keystone EC, Fish EN. Fibrocyte and T cell interactions promote disease pathogenesis in rheumatoid arthritis. J Autoimmun. 2016;69:38–50. doi: 10.1016/j.jaut.2016.02.008.
- Galligan CL, Siminovitch KA, Keystone EC, Bykerk V, Perez OD, Fish EN. Fibrocyte activation in rheumatoid arthritis. Rheumatology (Oxford) 2010;49:640–651. doi: 10.1093/rheumatology/kep265.
- Pilling D, Buckley CD, Salmon M, Gomer RH. Inhibition of fibrocyte differentiation by serum amyloid P. J Immunol. 2003;171:5537–5546. doi: 10.4049/jimmunol.171.10.5537.
- Marc G, Paer A, Pascale P, Mathieu D, Lila B, Arnaud M, Bruno C, Monique D, Christophe Q. A67 Acute lung Injury and Acute Respiratory distress syndrome: Biological mechanisms. American Thoracic Society: American Thoracic Society International Conference Abstracts; 2012. Differentiation of human blood Monocyte subsets into Fibrocytes is inhibited by alveolar environment during Acute lung Injury; p. A2113.
- Pilling D, Fan T, Huang D, Kaul B, Gomer RH. Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts. PLoS One. 2009;4:e7475. doi: 10.1371/journal.pone.0007475.
- Pilling D, Vakil V, Gomer RH. Improved serum-free culture conditions for the differentiation of human and murine fibrocytes. J Immunol Methods. 2009;351:62–70. doi: 10.1016/j.jim.2009.09.011.
- White MJ, Roife D, Gomer RH. Galectin-3 binding protein secreted by breast cancer cells inhibits Monocyte-derived Fibrocyte differentiation. J Immunol. 2015;195(4):1858–1867. doi: 10.4049/jimmunol.1500365.
- Shao DD, Suresh R, Vakil V, Gomer RH, Pilling D. Pivotal advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation. J Leukoc Biol. 2008;83:1323–1333. doi: 10.1189/jlb.1107782.
- Sciurba FC. Physiologic similarities and differences between COPD and asthma. Chest. 2004;126:117S–124S. doi: 10.1016/S0012-3692(15)31481-1.
- Zhang Y, Li H, Wu N, Dong X, Zheng Y. Retrospective study of the clinical characteristics and risk factors of rheumatoid arthritis-associated interstitial lung disease. Clin Rheumatol. 2017;36(4):817–823. doi: 10.1007/s10067-017-3561-5.
Source: PubMed