Scleroderma-specific autoantibodies embedded in immune complexes mediate endothelial damage: an early event in the pathogenesis of systemic sclerosis

• Published in: Arthritis research & therapy 2020-11, Vol.22 (1), p.1-265, Article 265
• Main Authors: Raschi, Elena, Privitera, Daniela, Bodio, Caterina, Lonati, Paola Adele, Borghi, Maria Orietta, Ingegnoli, Francesca, Meroni, Pier Luigi, Chighizola, Cecilia Beatrice
• Format: Article
• Language: English
• Subjects: Abnormalities; Antibodies; Antigens; Arthritis; Autoantibodies; Biopsy; Care and treatment; Cell culture; Deoxyribonucleic acid; Development and progression; Disease; DNA; Endothelial cells; Endothelium; Fibroblasts; Fibrosis; Immune complexes; Immune response; Lupus; Observations; Pathogenesis; Penicillin; Polyethylene glycol; RNA polymerase; Scleroderma; Scleroderma (Disease); Systemic sclerosis; Toll-like receptors; Tumor necrosis factor-TNF
• ISSN: 1478-6362; 1478-6354; 1478-6362
• DOI: 10.1186/s13075-020-02360-3

Background Consistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells. Methods ICs were purified from the sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase, and Th/To), patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS), or healthy controls (NHS) using polyethylene glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenI[alpha]1 (colI[alpha]1), interferon (IFN)-[alpha], and IFN-[beta] were investigated by real-time PCR; et-1 and il-6 mRNA levels were assessed after pre-treatment with bafilomycin. ICAM-1 expression was evaluated by cell ELISA; secretion of IL-6, IL-8, and transforming growth factor (TGF)-[beta]1 in culture supernatants was measured by ELISA. The expression of Fc[gamma] receptors (CD64, CD32, and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signaling pathways culminating with NFκB, p38MAPK, SAPK-JNK, and Akt were assessed by Western blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with ICs, and TGF-[beta]1 secretion and mRNA levels of colI[alpha]1 and matrix metalloproteinase (mmp)-1, protein expression of [alpha] smooth muscle actin ([alpha]-SMA), and IL-6 were evaluated by Western blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-[beta] inhibitors and stimulated with ATA-ICs. Results All SSc stimulated IL-6 secretion; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA-ICs and ARA-ICs upregulated et-1, and all SSc-ICs but ARA-ICs affected TGF-[beta]1 secretion. colI[alpha]1, IFN-[alpha], and IFN-[beta] mRNA levels were not affected by any SSc-IC. Fc[gamma]RII (CD32) and Fc[gamma]RIII (CD16) were not detectable on HUVECs, while Fc[gamma]RI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3, and tlr4 were upregulated by ATA-ICs and A