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

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 participate

The 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 publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Gating strategy for quantification of fibrocytes using lysed peripheral blood. Representative flow cytometry analysis showing the gating strategy. Population was initially gated on the basis of the forward side scatter characteristics and debris were eliminated, then lymphocyte and monocytes were selected, the CD45+ cells were gated in the plot “CD45-V500” versus forward scatter. Hereafter unwanted cells as B-Cells (CD19+), T-Cells (CD3+), basophils and eosinophils (CD294+) and dead cells (7AAD+) were removed using a PerCP-Cy5-5A dump channel. Finally, the CD45+ CD34+ Cd11b+ fibrocyte population was measured in the “CD11b-Pacific blue versus CD34-PE” plot. Fluoroscence minus one (FMO) controls are shown at the bottom

Fig. 2

Mature and circulating fibrocytes. Appearance of fibrocytes in a PBMCs cultured for 5 days in serum free medium; giemsa stain. b Cultured PBMCs. The arrow points at a pro-collagen type 1 positive cell. c Pro-collagen type 1 positive CD45+CD34+CD11b+ sorted cell from an RA patient. d Cultured fibroblast used as positive control for the pro-collagen type 1 stain. e IgG isotype control. The scale bar represents 100 μm

Fig. 3

Levels of CD45+ CD34+ CD11b+ cells and number of mature fibrocytes after 5 days in culture. a Comparison of CD45+ CD34+ CD11b+ cell levels. NS = not significant. b Comparison of number of mature fibrocytes after 5 days in culture. c Correlation between CD45+ CD34+ CD11b+ cells in blood measured by flow cytometry and number of mature fibrocytes after 5 days in culture

Fig. 4

Correlation of circulating fibrocytes with forced expiratory volume in 1 s (FEV1), in asthma patients

Fig. 5

Fibrocytes and diffusion capacity. a Comparison of circulating fibrocytes between RA patients with high or low hemoglobin corrected diffusion capacity. b Comparison of cultured mature fibrocytes between RA patients with high or low hemoglobin corrected diffusion capacity. c Comparison of circulating fibrocytes between all RA patients, RA patients with ILD included, with high or low hemoglobin corrected diffusion capacity. d Comparison of cultured mature fibrocytes between all RA patients, RA patients with ILD included, with high or low hemoglobin corrected diffusion capacity. e Correlation of circulating fibrocytes with hemoglobin corrected diffusion capacity in all RA patients including RA-ILD patients. f Correlation of cultured mature fibrocytes with hemoglobin corrected diffusion capacity in all RA patients, the RA patients with ILD included