Circulating Fibrocytes in Systemic Sclerosis Patients and Healthy Subjects: An in Vitro Study

Maurizio Cutolo¹, Paola Montagna², Stefano Soldano², Amelia Chiara Trombetta³, Paola Contini⁴, Vanessa Smith⁵, Barbara Ruaro², Alberto Sulli³ and Renata Brizzolara², ¹Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, IRCCS A.O.U. San Martino-IST, University of Genova, Genoa, Italy, Genoa, Italy, ²Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, IRCCS San Martino, Genoa, Italy, Genoa, Italy, ³Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, IRCCS A.O.U. San Martino-IST, University of Genova, Genoa, Italy, Genova, Italy, ⁴Division of Clinical Immunology, Department of Internal Medicine, University of Genova, Genoa, Italy, Genova, Italy, ⁵Faculty of Internal Medicine, Ghent University, Ghent, Belgium

Meeting: 2017 ACR/ARHP Annual Meeting

Date of first publication: September 18, 2017

Keywords: fibrosis and scleroderma

Session Information

Date: Sunday, November 5, 2017

Session Title: Systemic Sclerosis, Fibrosing Syndromes and Raynaud's – Pathogenesis, Animal Models and Genetics Poster I

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose:

Circulating fibrocytes seem to exert immunomodulatory effects, expressing class II major histocompatability complex molecules (HLA-DP, -DQ, and -DR) and could have a role in fibrosing diseases (i.e. systemic sclerosis, SSc), due to the capacity of such cells to migrate into affected tissues (through CXCR4/CXCL12 interaction) and to differentiate into fibroblasts/myofibroblasts, then inducing matrix protein deposition and fibrosis [1-4].

The aim of this study was to isolate fibrocytes from peripheral blood mononuclear cells (PBMCs) of SSc patients and healthy subjects (CNTs) and comparing both after having identified them by fluorescence-activated cell sorter analysis (FACS) (using their specific markers: the leukocyte common antigen CD45, collagen I (COL I), the chemokine receptor CXCR4 and HLA-DR [2]).

Methods:

Blood samples were collected, after signed informed consent, at basal time (T0), from 11 SSc patients (treated only with different vasodilator drugs) and 5 CNTs. In addition, PBMCs, isolated from 9 SSc patients and 5 CNTs, were cultured on fibronectin-coated plates [5]. The non-adherent cells were removed and after 8 days (T8) of culture (standardized time) the adherent spindle shaped cells were lifted through incubation in 0.05% EDTA (ice-cold). Fibrocyte identification was performed by FACS, using anti-CD45, anti-COL I, anti-CXCR4 and anti-HLA-DR monoclonal antibodies.

Results:

FACS analysis revealed that, at T0, among the CD45+ cells, the percentage of fibrocytes, identified as CD45+, COL I+, CXCR4+ was 1.0±1.2 % in SSc patients and 0.5±0.2 % in healthy subjects (CNTs). In addition, the HLA-DR expression on fibrocytes in both CNTs and SSc patients showed low values (22.1±21.1 % and 13.1±4.7 %, respectively).

After 8 days (T8) of culture, fibrocytes presented adherent and spindle shaped morphology and FACS analysis demonstrated that the percentage of fibrocytes CD45+, COL I+, CXCR4+ increased up to 52.8±27.1% in SSc patients and up to 61.9±24.4 in CNTs, compared to T0.

Therefore, at T8 of culture, the HLA-DR+ expression on fibrocytes in SSc patients and CNTs strongly increased (90.1±22.7 % and 97.9±1.9, respectively), compared to T0.

Conclusion:

Circulating fibrocytes, identified as CD45, COL I and CXCR4 positive cells, resulted in very low percentage in peripheral blood at basal time, but after 8 days of culture in proper conditions, the percentage of differentiated fibrocytes increased up to 50 times in SSc (and CNTs), whereas the HLA-DR expression increased up to 68% in SSc and 85% in CNTs. Data suggest that circulating fibrocytes might be an early cellular targets in presence of fibrositing diseases.

References:

1. Blakaj and Bucala. Fibrogenesis & Tissue Repair 2012;5:S6. 2. Chesney J et al. Proc. Natl. Acad. Sci. 1997; 94:6307–6312. 3. Bucala R. Mol Med 2015;21:S3–S5. 4. Brunasso A.M. et al. F1000Research 2016;5:723. 5. Pilling D et al. J Immunol Methods 2009; 351:62–70.

Disclosure: M. Cutolo, None; P. Montagna, None; S. Soldano, None; A. C. Trombetta, None; P. Contini, None; V. Smith, None; B. Ruaro, None; A. Sulli, None; R. Brizzolara, None.

To cite this abstract in AMA style:

Cutolo M, Montagna P, Soldano S, Trombetta AC, Contini P, Smith V, Ruaro B, Sulli A, Brizzolara R. Circulating Fibrocytes in Systemic Sclerosis Patients and Healthy Subjects: An in Vitro Study [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/circulating-fibrocytes-in-systemic-sclerosis-patients-and-healthy-subjects-an-in-vitro-study/. Accessed April 10, 2020.

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