Antifibroblast antibodies in systemic sclerosis induce fibroblasts to produce profibrotic chemokines, with partial exploitation of toll-like receptor 4.
OBJECTIVE
Previous studies have revealed the presence of IgG antifibroblast antibodies (AFAs) capable of binding to the surface of fibroblasts in systemic sclerosis (SSc) sera. Since chemokines may directly or indirectly affect the development of fibrosis, this study was undertaken to investigate the production of chemokines induced by AFAs in fibroblasts, and to characterize the signaling pathways and surface molecules involved.


METHODS
AFA-positive and AFA-negative IgG were tested on fibroblasts. Chemokine messenger RNA expression was screened by microarray and quantitative reverse transcription-polymerase chain reaction. Production of CCL2, CXCL8, and CXCL10 proteins was assessed by enzyme-linked immunosorbent assay. Pharmacologic inhibitors were used to study signal transduction, with results assessed by Western blotting and immunofluorescence analysis. Fibroblasts with defective expression of Toll-like receptors (TLRs) and anti-TLR monoclonal antibodies (mAb) were used to assess AFA specificity.


RESULTS
In human fibroblasts, AFA-positive IgG induced the preferential transcription of chemokines with profibrotic and proangiogenic potential, including, but not exclusively, CCL2, CXCL1, CXCL8, CKLF, and ECGF1, which were distinctly different from those induced by interferon-gamma. Levels of CCL2 and CXCL8 proteins were increased in AFA-stimulated fibroblast culture supernatants. AFA binding to fibroblasts resulted in concomitant activation of ERK-1/2, c-Jun, and NF-kappaB. CCL2 production was sensitive to inhibition of both proteasome and JNK, while CXCL8 production was sensitive only to inhibition of proteasome. AFAs failed to up-regulate CCL2 expression in TLR-4-deficient fibroblasts but not in TLR-6- or TLR-2-deficient fibroblasts. Moreover, anti-TLR-4 mAb, but not anti-TLR-2 mAb, partially inhibited the production of CCL2 induced by AFAs in human fibroblasts.


CONCLUSIONS
Autoantibodies that bind to the surface of fibroblasts may contribute to the pathogenesis of SSc by up-regulating the fibroblast production of profibrotic and proangiogenic chemokines, in a proteasome- and TLR-4-dependent manner.
Fineschi
Serena
S
University Hospital and School of Medicine, Geneva, Switzerland.
Goffin
Laurence
L
Rezzonico
Roger
R
Cozzi
Franco
F
Dayer
Jean-Michel
JM
Meroni
Pier Luigi
PL
Chizzolini
Carlo
C
D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000097:blood*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics; D054357:Chemokine CXCL10 / Q000235:genetics; D018925:Chemokines / Q000235:genetics; D020405:Dermis / Q000166:cytology; D005260:Female; D005347:Fibroblasts / Q000276:immunology* / Q000473:pathology; D005355:Fibrosis; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D016209:Interleukin-8 / Q000235:genetics; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism*; D015533:Transcriptional Activation / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; C111835:CCL2 protein, human; C114753:CXCL10 protein, human; C508606:CXCL8 protein, human; D018932:Chemokine CCL2; D054357:Chemokine CXCL10; D018925:Chemokines; D007074:Immunoglobulin G; D016209:Interleukin-8; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4