Antifibroblast antibodies from systemic sclerosis patients are internalized by fibroblasts via a caveolin-linked pathway.
OBJECTIVE
Fibroblast activation is a crucial event in the development of systemic sclerosis (SSc). Antifibroblast autoantibodies (AFAs), detectable in the sera of SSc patients, are able to induce a proinflammatory phenotype on cultured fibroblasts. This study was undertaken to investigate the mechanisms of the interaction between AFAs and living fibroblasts.


METHODS
We coupled to fluorescein 1) IgG purified from AFA-positive and AFA-negative SSc sera (as assessed by cellular enzyme-linked immunosorbent assay) and 2) single healthy donor and pooled normal IgG. The interaction of IgG with living cultured fibroblasts from healthy individuals and from a patient with SSc was visualized by real-time confocal microscopy. Intracellular colocalization of caveolin and internalized AFA-positive IgG was assessed by immunofluorescence.


RESULTS
AFA-positive IgG bound to living fibroblasts and was internalized with a cytoplasmic fibrillar pattern, in contrast to AFA-negative IgG. In the IgG tested, no correlation with antinuclear antibody activity was found. Preincubation of fibroblasts with normal IgG did not affect internalization. Internalized AFA-positive IgG colocalized with caveolin, and internalization was entirely inhibited by disassembling fibroblast caveolae with filipin.


CONCLUSIONS
The finding that both normal and pathologic fibroblasts specifically internalized AFA-positive, but not AFA-negative, IgG demonstrates that AFAs in SSc patient sera interact with constitutively expressed membrane molecules on fibroblasts, via an Fc-independent mechanism. The results of colocalization and inhibition experiments suggest that microdomains containing caveolin are involved in the interaction between AFAs and fibroblasts. These data, together with the reported ability of AFAs to activate fibroblasts, provide evidence for a role of AFAs in the pathogenesis of SSc.
Ronda
Nicoletta
N
Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Via Gramsci 14, 43100 Parma, Italy. nicoletta.ronda@unipr.it
Gatti
Rita
R
Giacosa
Roberto
R
Raschi
Elena
E
Testoni
Cinzia
C
Meroni
Pier Luigi
PL
Buzio
Carlo
C
Orlandini
Guido
G
D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000378:metabolism* / Q000494:pharmacology; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured; D003287:Contrast Media / Q000493:pharmacokinetics; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D019793:Fluorescein / Q000493:pharmacokinetics; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D012595:Scleroderma, Systemic / Q000276:immunology* / Q000378:metabolism
D001323:Autoantibodies; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003287:Contrast Media; D007074:Immunoglobulin G; D019793:Fluorescein