Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

Abstract

Background: In systemic sclerosis (SSc), autoantibodies provide the most accurate tool to predict the disease subset and pattern of organ involvement. Scleroderma autoantibodies target nucleic acids or DNA/RNA-binding proteins, thus SSc immune complexes (ICs) can embed nucleic acids. Our working hypothesis envisaged that ICs containing scleroderma-specific autoantibodies might elicit proinflammatory and profibrotic effects in skin fibroblasts.

Methods: Fibroblasts were isolated from skin biopsies obtained from healthy subjects and patients with diffuse cutaneous SSc (dcSSc). ICs were purified by polyethylene-glycol precipitation from sera of SSc patients bearing different autoantibodies. ICs from patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS) and from normal healthy subjects (NHS) were used as controls. After incubation with ICs, fibroblasts were evaluated for ICAM-1 expression, interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase (MMP)-2, tumor growth factor (TGF)-β1 and Pro-CollagenIα1 secretion, collagen (col)Iα1, mmp-1, toll-like receptor (tlr)2, tlr3, tlr4, tlr7, tlr8, tlr9, interferon (ifn)-α, ifn-β and endothelin-1 mRNA, and NFκB, p38MAPK and SAPK-JNK activation rate. Experiments were also performed after pretreatment with DNase I/RNase and NFκB/p38MAPK inhibitors.

Results: The antigenic reactivity for each SSc-IC mirrored the corresponding serum autoantibody specificity, while no positivity was observed in NHS-ICs or sera. SSc-ICs but not NHS-ICs increased ICAM-1 expression, stimulated IL-6, IL-8, MMP-2, MCP-1, TGF-β1 and Pro-CollagenIα1 secretion, upregulated et-1, ifn-α, ifn-β, tlr2, tlr3 and tlr4, and activated NFκB, p38MAPK and SAPK-JNK. tlr9 was significantly upregulated by ARA-ICs, mmp-1 was significantly induced by ACA-ICs whereas colIα1 was not modulated by any SSc-ICs. SLE-ICs and PAPS-ICs significantly upregulated MMP-2 and activated NFκB, p38MAPK and SAPK-JNK. SLE-ICs and PAPS-ICs did not affect colIα1, mmp-1 and Pro-CollagenIα1. DNase I and RNase treatment significantly reduced the upregulation of study mediators induced by SSc-ICs. Pretreatment with NFκB/p38MAPK inhibitors suggested that response to anti-Th/To-ICs was preferentially mediated by p38MAPK whereas ATA-ICs, ACA-ICs and ARA-ICs engaged both mediators. In dcSSc fibroblasts, stimulation with SSc-ICs and NHS-ICs upregulated IL-6 and IL-8.

Conclusions: These data provide the first demonstration of the proinflammatory and profibrotic effects of SSc-ICs on fibroblasts, suggesting the potential pathogenicity of SSc autoantibodies. These effects might be mediated by Toll-like receptors via the interaction with nucleic acid fragments embedded in SSc-ICs.

Keywords: Autoantibodies; Fibroblasts; Fibrosis; Immune complexes; Inflammation; Systemic sclerosis; Toll-like receptors.

Fig. 1

TaqMan® Gene Expression assays against SSc-specific antigens of PEG-precipitated ICs and corresponding sera evaluated by EUROLINE-SSc profile kit. One ATA-IC and one NHS-IC presented as representative assay. CTR+, assay-positive control. a, Ro-52; b, PDGF receptor; c, Ku; d, PM-Scl75; e, PM-Scl100; f, Th/To; g, NOR90; h, Fibrillarin; i, RP155; l, RP11; m, CENP B; n, CENP A; o, Scl-70 (DNA topoisomerase I). ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects

Fig. 2

Dose–response dilution curve for ICAM-1 expression on fibroblast cell surface. Fibroblasts exposed to serial two-fold dilutions (from 1:2 to 1:64) of SSc-ICs and NHS-ICs, and ICAM-1 evaluated by cell ELISA. anti-Th/To anti-Th/To antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects, OD optical density

Fig. 3

ICAM-1 expression on fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, NHS normal healthy subjects, OD optical density, poly(I:C) polyinosinic-polycytidylic acid

Fig. 4

IL-6, IL-8, MMP-2 and MCP-1 levels in culture supernatants from fibroblasts incubated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8; c MMP-2; d MCP-1. **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, IL interleukin, LPS lipopolysaccharide, MCP monocyte chemoattractant protein, MMP matrix metalloproteinase, NHS normal healthy subjects, poly(I:C) polyinosinic-polycytidylic acid

Fig. 5

et-1, ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1; b ifn-α; c ifn-β. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, et-1 endothelin-1, IFN interferon, IC immune complex, LPS lipopolysaccharide, NHS normal healthy subjects, poly(I:C) polyinosinic-polycytidylic acid

Fig. 6

TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1; d mmp-1. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, colIα1 collagenIα1, IC immune complex, MMP matrix metalloproteinase, NHS normal healthy subjects, TGF tumor growth factor

Fig. 7

tlr mRNA expression levels in fibroblasts stimulated with SSc-ICs or control NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a tlr2; b tlr3; c tlr4; d tlr9. **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, NHS normal healthy subjects, poly(I:C) polyinosinic-polycytidylic acid, TLR Toll-like receptor

Fig. 8

Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, MAPK mitogen activated kinase, NHS normal healthy subjects, NFκB nuclear factor kappa B, pNFκB phosphorylated NFκB, pp38MAPK phosphorylated p38MAPK, pp54SAPK-JNK phosphorylated p54SAPK-JNK, pp46SAPK-JNK phosphorylated p46SAPK-JNK

Fig. 9

Intracellular signaling pathways in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to SLE-ICs, PAPS-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. IC immune complex, LPS lipopolysaccharide, MAPK mitogen activated kinase, NHS normal healthy subjects, NFκB nuclear factor kappa B, pNFκB phosphorylated NFκB, pp38MAPK phosphorylated p38MAPK, pp54SAPK-JNK phosphorylated p54SAPK-JNK, pp46SAPK-JNK phosphorylated p46SAPK-JNK, PAPS primary anti-phospholipid syndrome, SLE systemic lupus erythematosus

Fig. 10

colIα1 and mmp-1 mRNA expression and Pro-CollagenIα1 and MMP-2 secretion in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to PAPS-ICs, SLE-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) and LPS (1 μg/ml) used as positive control for collagen synthesis and secretion. a colIα1; b mmp-1; c Pro-CollagenIα1; d MMP-2. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. colIα1 collagenIα1, IC immune complex, LPS lipopolysaccharide, MMP matrix metalloproteinase, NHS normal healthy subjects, PAPS primary anti-phospholipid syndrome, SLE systemic lupus erythematosus, TGF tumor growth factor

Fig. 11

et-1, tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2; c ATA-ICs and anti-Th/To-ICs on ifn-α; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, et-1 endothelin-1, IC immune complex, IFN interferon, TLR Toll-like receptor

Fig. 12

Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated (a) NFκB and (b) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). *p < 0.01, **p < 0.001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, NFκB nuclear factor kappa B, NHS normal healthy subjects, MAPK mitogen activated kinase, pp38MAPK phosphorylated p38MAPK

Fig. 13

TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, IL interleukin, LPS lipopolysaccharide, NFκB nuclear factor kappa B, NHS normal healthy subjects, TGF tumor growth factor

Fig. 14

TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, IL interleukin, LPS lipopolysaccharide, NHS normal healthy subjects, MAPK p38 mitogen activated kinase, TGF tumor growth factor

Fig. 15

IL-6 and IL-8 in culture supernatants from dcSSc fibroblasts incubated with SSc-ICs or NHS-ICs. dcSSc fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8. *p < 0.01, **p < 0.001, ***p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, IL interleukin, LPS lipopolysaccharide, NHS normal healthy subjects, poly(I:C) polyinosinic-polycytidylic acid