08.25 In vitro characterisation of circulating fibrocytes from systemic sclerosis patients with diffuse disease: early results

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Abstract

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).

Background

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).

Results

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 Sci1997;94:6307–6312;

References

2. Herzoga EL, et al. Experimental Haematology2010;38:548–556.

References

3. Brunasso A.M. et al. F1000Research2016;5:723.

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