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?

Corallo, Claudio; Cheleschi, Sara; Cutolo, Maurizio; Soldano, Stefano; Fioravanti, Antonella; Volpi, Nila; Franci, Daniela; Nuti, Ranuccio; Giordano, Nicola Giuseppe

doi:10.1186/s13075-019-1931-x

Cited by 18 publications

(12 citation statements)

References 49 publications

(49 reference statements)

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“…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 ].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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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), collagenIα1 (colIα1), interferon (IFN)-α, and IFN-β 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)-β1 in culture supernatants was measured by ELISA. The expression of Fcγ 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-β1 secretion and mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of α smooth muscle actin (α-SMA), and IL-6 were evaluated by Western blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-β 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-β1 secretion. colIα1, IFN-α, and IFN-β mRNA levels were not affected by any SSc-IC. FcγRII (CD32) and FcγRIII (CD16) were not detectable on HUVECs, while FcγRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3, and tlr4 were upregulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 upregulation. Pre-treatment with bafilomycin did not affect the upregulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs, and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and Akt, and all SSc-ICs but ARA-ICs yielded the activation of NFκB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK. When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-β1 secretion, colIα1, α-SMA, and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-β inhibitors prevented et-1 upregulation induced by ATA-ICs by 85% and 77%, respectively. Conclusions These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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Objectives1) To detect functionally active antibodies(abs) to the angiotensin II type-1-receptor (AT1R) by a novel luminometric assay. 2) To assess their prevalence in systemic sclerosis (SSc), other collagen disorders, as well as in further chronic inflammatory disorders including autoimmune, toxic and chronic viral diseases. 3) To compare these abs with anti-AT1R antibodies by ELISA as well as with antibodies to endothelin-type-A receptors (ETA1) and to topoisomerase I (topo-I) with respect to their specificity and clinical relevance.MethodsSera from 98 SSc-patients, 110 patients with other chronic inflammatory rheumatic disorders, 97 patients with autoimmune liver diseases, 57 patients with toxic or chronic viral liver diseases and 36 healthy controls were analyzed. A luminometric bioassay was established with Huh-7-cells constitutively expressing the AT1R. Patients’ sera were also tested by commercially available ELISA for anti-AT1R, -ETA1- and by an in-house ELISA for anti–topo-I-abs.ResultsFifty-two percent of the SSc-patients had functionally active anti-AT1R-abs with stimulatory (34%) or inhibitory capacity (18%). They were present also in up to 59% of patients with other rheumatic diseases but only 22% of healthy individuals (sensitivity 52%, specificity 53%). The functionally active antibodies detected by the luminometric assay did not correlate with anti-AT1R-, -ETA1- or -topo-I-abs measured by ELISA, but there was a strong correlation between anti-topo-I-, AT1R-, and -ETA1-ab reactivity measured by ELISA. Sensitivities of 55%, 28% and 47% and specificities of 66%, 87%, and 99% were calculated for these anti-AT1R-, -ETA1-, and anti-topo-I-abs, respectively. Functionally active abs did not correlate with disease severity or any organ manifestation. In contrast, abs to topo-I, AT1R, and ETA1 were associated with digital ulcers, pulmonary- and esophageal manifestation.ConclusionsFunctionally active anti-AT1R-abs can be detected in SSc-patients but do not correlate with disease activity. They are not specific for this disease and occur also in other autoimmune disorders and even viral or toxic diseases. Also, the vascular antibodies detected by ELISA are not SSc-specific but correlated with disease manifestations. In contrast, anti-topo-I-abs were confirmed to be a highly specific biomarker for both, diagnosis and organ manifestations of SSc.

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“…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].…”

Section: Discussionmentioning

confidence: 99%

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

Raschi

Privitera

Bodio

et al. 2020

Preprint

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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 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), primary anti-phospholipid syndrome (PAPS) or healthy controls (NHS) using polyethylen glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenIα1 (colIa1), interferon (IFN)-α and IFN-β 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)-β1 in culture supernatants was measured by ELISA. The expression of Fcg receptors (CD64, CD32 and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signalling pathways culminating with NFkB, p38MAPK, SAPK-JNK and Akt were assessed by Western Blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVEC incubated with ICs, and TGF-b1 secretion, mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of . a smooth muscle actin (a-SMA) and IL-6 were evaluated by Western Blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-b 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 and ARA-ICs up-regulated et-1 and all SSc-ICs but ARA-ICs affected TGF-b1 secretion. colIa1, IFN-a and IFN-b mRNA levels were not affected by any SSc-ICs. FcgRII (CD32) and FcgRIII (CD16) were not detectable on HUVECs, while FcgRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3 and tlr4 were up-regulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 up-regulation. Pre-treatment with bafilomycin did not affect the up-regulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and AKT, all SSc-ICs but ARA-ICs yielded the activation of NFkB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK.When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-b1 secretion, colIa1, a-SMA and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-b inhibitors prevented et-1 up-regulation induced by ATA-ICs by 85% and 77% respectively.Conclusions: These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

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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?

Cited by 18 publications

References 49 publications

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

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

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

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

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