Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin

Abstract

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Figures

Figure 1. ACPAs are linked to high bone resorption in humans.

Serum samples obtained from healthy controls as well as patients with RA without ACPAs or with positivity for rheumatoid factor (RF) or ACPAs were investigated for the bone resorption parameters CTXI (A and B), TRAP5b (C), and cathepsin K (D). In B, different ACPA responses are shown (expressed in units). *P < 0.05; **P < 0.01.

Figure 2. Isolation of ACPAs from human serum.

(A) ACPA reactivity of the original serum from RA patients, purified ACPAs, the corresponding Fab fragment (ACPA/Fab), and the remaining eluated IgG fraction (eluate) for 3 individual serum samples as well as the pooled samples. Dotted curves in the graph of the pooled sample indicate detection with an Fc- specific antibody, with loss of reactivity of the Fab fraction confirming its purity. (B) Coomassie gel and Western blot of HeLa cells showing the binding of purified antibodies against vimentin (VIM), MCV, and cytosolic (1), membrane (2), nuclear (3) and actin-containing cytoskeletal (4) cell factions before (–) and after (+) treatment with PAD. (C) ELISA showing reactivity of ACPAs and MCV-ACPAs against noncitrullinated and citrullinated peptides of vimentin, fibrinogen, and CTXI. y axis shows the binding ratio with specificity to citrullinated peptides giving values higher than 1 (dotted line). (D) Sialylation of MCV-ACPAs was analyzed by assessing the S2 glycoform (anti-sial ACPA) by ELISA before and after neuraminidase (NA) treatment. (E) Epitope reaction pattern of MCV-ACPAs was analyzed by assessing binding to 25-mer peptides spanning the entire sequence of citrullinated vimentin. OD410nm, OD at a wavelength of 410 nm. *P < 0.05.

Figure 3. Expression of PAD enzyme and citrullinated vimentin in osteoclasts.

(A and B) Real-time PCR for PAD2 (A) and PAD4 (B) of human osteoclast precursor cells stimulated with MCSF and RANKL to achieve osteoclast differentiation. (C) Western blotting showing protein expression of PAD4 and PAD2 at various stages of osteoclast differentiation. (D) Western blotting showing protein expression of vimentin at various stages of osteoclast differentiation (E and F) Western blotting showing MCV expression using a chicken antibody against citrullinated vimentin (E; recombinant vimentin used as positive control) and MCV-ACPAs (F). For control purposes, staining for β-actin was performed. Representative blots from 3 independent experiments are shown.

Figure 4. MCV-ACPAs stimulate osteoclastogenesis.

(A) Laser scanning microscopy of 2-mm osteoclast sections showing binding of MCV-ACPAs. Left: green, MCV-ACPA staining for citrullinated vimentin; red, phalloidin staining for actin; blue, DAPI staining for the nucleus. Right: green, vimentin staining; red, MCV-ACPA staining for citrullinated vimentin; blue, DAPI staining for the nucleus. Arrowheads indicate surface staining for citrullinated vimentin. (B) Osteoclastogenesis (n = 3) induced by 10 ng/ml MCSF, 1 ng/ml RANKL, and different concentrations of MCV-ACPAs and of IgG fractions deprived of ACPAs (eluate) in the presence of MCSF and RANKL. N.Oc, number of osteoclasts. (C) Resorption pit assay (n = 3) with different concentrations of MCV-ACPAs and of IgG fractions deprived of ACPA. (D) Assessment of resorption index (n = 3) by transposing a defined number of osteoclasts on bone slices and measuring the resorption pit size per single osteoclast. Control indicates no antibody addition. Original magnification, ×20 (A); ×100 (A, enlarged view); ×10 (B and D). Scale bars: 100 μm. *P < 0.05.

Figure 5. MCV-ACPAs induce bone loss in vivo.

(A) 3-dimensional (top) and 2-dimensional (bottom) μCT images of the tibial metaphysis of Rag1–/– mice that were left untreated or treated with control IgG or MCV-ACPAs. (B) Structural parameters of the tibial bone (n = 5 per group), including the ratio of bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and connectivity density (Conn.D). (C) Microphotographs (original magnification, ×400) of tibial bones of Rag1–/– mice treated with either IgG or MCV-ACPAs, stained for osteoclasts (purple stain and arrows) by histochemical detection of TRAP. Histomorphometric quantification of osteoclast number and osteoblast number (N.Ob) on the tibial bone surface (BS) is also shown. *P < 0.05.

Figure 6. Bone loss elicited by MCV-ACPAs is induced by TNF-mediated increase of osteoclast precursor trafficking and differentiation.

(A) Serum levels (n = 5 per group) of CTXI, osteocalcin, and TNF-α in Rag1–/– mice that were left untreated or treated with control IgG or MCV-ACPAs. (B) FACS analysis (n = 5 per group) of spleen cells for the number of CD11b+CD14+ cells (osteoclast precursors) and their expression of CD115 and RANK. (C) Osteoclastogenesis assay (n = 3 per group) of spleen cells from Rag1–/– mice treated with control IgG or MCV-ACPAs with various concentrations of RANKL. TNF-α release and osteoclastogenesis assays (n = 3 per group) from spleen cells of wild-type mice treated with control IgG or MCV-ACPAs are also shown. aTNF, blockade of TNF-α by 100 ng/ml etanercept. *P < 0.05.