Abstract
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 (ECs).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 ECs (HUVECs) were incubated with ICs, IL-1β, Poly I:C, LPS or ODN CpG. mRNA levels of endothelin-1 (ET-1), collagenIα1 (colIα1), interferon (IFN)-α and IFN-β were investigated by Real-Time PCR; ICAM-1 expression was evaluated by cell-ELISA; secretion of IL-6, IL-8 and tumour growth factor (TGF)-β1 in culture supernatants was measured by ELISA. 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 tumour growth factor (TGF)-β1secretion, mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1 were evaluated.Results: All SSc stimulated IL-6 secretion compared to NHS-ICs; 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-β1 secretion. ColIα1, IFN-α and IFN-β mRNA levels were not affected by any SSc-ICs. 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 up-regulation. All SSc-ICs activated p38MAPK and AKT, 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 and colIα1 expression levels were significantly modulated.Conclusions: These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies in ECs. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.
Immunohistological analysis of tumour growth factor beta 1 expression in normal and inflamed salivary glands.
