Antibodies against specific extractable nuclear antigens (ENAs) as diagnostic and prognostic tools and inducers of a profibrotic phenotype in cultured human skin fibroblasts: are they functional?

Abstract

Background: The importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become recognized by their incorporation into the 2013 ACR/EULAR classification criteria. Clear prognostic and phenotypic associations with cutaneous subtype and internal organ involvement have been also described. However, little is known about the potential of autoantibodies to exert a direct pathogenic role in SSc. The aim of the study is to assess the pathogenic capacity of anti-DNA-topoisomerase I (anti-Topo-I) and anti-centromeric protein B (anti-Cenp-B) autoantibodies to induce pro-fibrotic markers in dermal fibroblasts. Methods: Dermal fibroblasts were isolated from unaffected and affected skin samples of (n = 10) limited cutaneous SSc (LcSSc) patients, from affected skin samples of diffuse cutaneous (DcSSc) patients (n = 10) and from healthy subjects (n = 20). Fibroblasts were stimulated with anti-Topo-I, anti-Cenp-B IgGs, and control IgGs in ratios 1:100 and 1:200 for 24 h. Cells were also incubated with 10% SSc anti-Topo-I + and anti-Cenp-B + whole serum and with 10% control serum for 24 h. Viability was assessed by MTT test, while apoptosis was assessed by flow cytometry. Activation of pro-fibrotic genes ACTA2, COL1A1, and TAGLN was evaluated by quantitative real-time PCR (qPCR), while the respective protein levels alpha-smooth-muscle actin (α-SMA), type-I-collagen (Col-I), and transgelin (SM22) were assessed by immunocytochemistry (ICC). Results: MTT showed that anti-Cenp-B/anti-Topo-I IgGs and anti-Cenp-B + /anti-Topo-I + sera reduced viability (in a dilutiondependent manner for IgGs) for all the fibroblast populations. Apoptosis is induced in unaffected LcSSc and control fibroblasts, while affected LcSSc/DcSSc fibroblasts showed apoptosis resistance. Basal mRNA (ACTA2, COL1A1, and TAGLN) and protein (α-SMA, Col-1, and SM22) levels were higher in affected LcSSc/DcSSc fibroblasts compared to LcSSc unaffected and to control ones. Stimulation with anti-Cenp-B/anti-Topo-I IgGs and with anti-Cenp-B + /anti-Topo-I + sera showed a better induction in unaffected LcSSc and control fibroblasts. However, a statistically significant increase of all pro-fibrotic markers is reported also in affected LcSSc/DcSSc fibroblasts upon stimulation with both IgGs and sera. Conclusions: This study suggests a pathogenic role of SSc-specific autoantibodies to directly induce pro-fibrotic activation in human dermal fibroblasts. Therefore, besides the diagnostic and prognostic use of those autoantibodies, these data might further justify the importance of immunosuppressive drugs in the early stages of the autoimmune disease, including SSc.

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

“…In particular, it has been very recently documented that sera containing ATA and ACA as well as polyclonal IgG antibodies targeting DNA topoisomerase I and centromeric protein B affect the viability and apoptosis rate of healthy skin fibroblasts. The same treatments upregulated pro-fibrotic molecules as α-SMA, colIα1, and transgelin, assessed both as mRNA expression and as protein levels at immunochemistry [ 34 ]. To overcome criticisms advocating the nuclear localization of both antigens, the authors claimed that, according to previous studies, DNA topoisomerase I and centromeric protein B can be released from damaged endothelial cells [ 35 , 36 ].…”

Functionally Active Antibodies to the Angiotensin II Type 1-Receptor Measured by a Luminometric Bioassay Do Not Correlate With Clinical Manifestations in Systemic Sclerosis: A Comparison With Antibodies to Vascular Receptors and Topoisomerase I Detected by ELISA

“…Anti-topo-I antibodies are more prevalent inbut not restricted to -diffuse cutaneous (dc) SSc whereas ACA are more frequent in limited cutaneous (lc) SSc (2)(3)(4). They are useful markers for diagnosis and prognosis of organ involvement but their contribution to disease pathogenesis is still under investigation (2,5,6).…”

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

“…In particular, it has been very recently documented that sera containing ATA and ACA as well as polyclonal IgG antibodies targeting DNA topoisomerase I and centromeric protein B affect the viability and apoptosis rate of healthy skin broblasts. The same treatments up-regulated pro-brotic molecules as a-SMA, colIa1 and transgelin, assessed both as mRNA expression and as protein levels at immunochemistry [33]. To overcome criticisms advocating the nuclear localization of both antigens, the authors claimed that, according to previous studies, DNA topoisomerase I and centromeric protein B can be released from damaged endothelial cells [34,35].…”