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

<strong>Background: </strong>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. <strong>Methods: </strong>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 <em>endothelin-1 (et-1)</em>, <em>collagenIα1 (colI</em><em>a</em><em>1)</em>, <em>interferon (IFN)-α</em> and <em>IFN-β</em> were investigated by Real-Time PCR; <em>et-1</em> and <em>il-6</em> 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 <em>colIα1</em> and <em>matrix metalloproteinase (mmp)-1</em>, protein expression of a smooth muscle actin (a-SMA) and IL-6 were evaluated by Western Blotting<em>; et-1</em> mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-b inhibitors and stimulated with ATA-ICs. <strong>Results: </strong>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 <em>et-1 </em>and all SSc-ICs but ARA-ICs affected TGF-b1 secretion. c<em>olI</em><em>a</em><em>1</em>, <em>IFN-</em><em>a</em> and <em>IFN-</em><em>b </em>mRNA levels were not affected by any SSc-IC. FcgRII (CD32) and FcgRIII (CD16) were not detectable on HUVECs, while FcgRI (CD64) was minimally expressed. A differential modulation of<em> tlr</em> expression was observed: <em>tlr2</em>, <em>tlr3</em> and <em>tlr4</em> were up-regulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in <em>tlr9</em> up-regulation. Pre-treatment with bafilomycin did not affect the up-regulation of <em>et-1</em> and <em>il-6</em> 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, <em>colI</em><em>a</em><em>1, </em>a-SMA and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-b inhibitors prevented <em>et-1</em> up-regulation induced by ATA-ICs by 85% and 77% respectively. <strong>Conclusions: </strong>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.