Background Circulating fibrocytes seem an important source of fibroblasts/myofibroblasts in physiological and pathological tissue remodelling, such as in tissue repair and in fibrosing diseases (ie, systemic sclerosis, SSc).^1–3 In addition, fibrocytes seem to express class II major histocompatability complex molecules (HLA-DP, -DQ, and -DR).

We aimed to isolate fibrocytes from peripheral blood mononuclear cells (PBMCs) of healthy subjects and SSc patients and to identify them using their specific markers: CD45, HLA-DR, collagen I, and the chemokine receptor CXCR4.²

Materials and methods PBMCs were collected, at basal time (t0), from 5 healthy subjects (CNTs) and 9 SSc patients, affected by diffuse disease (dSSc) and with treatment limited to vasodilators (prostacyclin) and aspirin. PBMCs isolated from 7 among the SSc patients were cultured on fibronectin-coated plates. The non-adherent cells were removed and after 8 days (t8) of culture (standardised time), the adherent cells were lifted through incubation in 0.05% EDTA (ice-cold). Fibrocyte identification (at both t0, t8), was done by fluorescence-activated cell sorter analysis (FACS), using anti-CD45, anti-collagen type I, anti-CXCR4 and HLA-DR monoclonal antibodies.

Results At basal time (t0), among the CD45+ cells, the percentage of fibrocytes, identified as triple positive (CD45+, coll I+, CXCR4+), resulted 0.44%±0.31% in healthy subjects (CNTs) and 2.10%±2.6% in SSc patients. In addition, the HLA-DR expression on fibrocytes in both CNTs and SSc patients showed low values (7.06%±6.1% and 24.2%±22.2%, respectively).

Interestingly, after 8 days (t8) of culture, fibrocytes CD45+, coll I+, CXCR4+ from the SSc patients increased up to 45.88%±23.71%, compared to basal time (t0). In addition, at t8, fibrocytes from SSc patients strongly increased HLA-DR+ expression (88.2%±24.8%).

Conclusion Fibrocytes isolated from circulating progenitor cells (CPCs) of healthy subjects and SSc patients were confirmed to express CD45, coll I and CXCR4 molecules, but in very low percentage. Already after 8 days of culture, fibrocytes (CD45+, coll I+ and CXCR4+) from SSc patients, increased up to 48% and the HLA-DR expression increased up to 64%. Additional markers for these CPCs are now under test to further characterise their phenotype(s) vs healthy controls.

References 1. Chesney J, et al. Proc Natl Acad Sci 1997;94:6307–6312;

2. Herzoga EL, et al. Experimental Haematology 2010;38:548–556.

3. Brunasso A.M. et al. F1000Research 2016;5:723.