IgA Immune Complexes Induce Osteoclast-Mediated Bone Resorption

Abstract

Objective: Autoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.

Methods: Anti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption.

Results: NET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts.

Conclusion: IgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.

Keywords: ACPA; IgA; autoantibodies; bone resorption; osteoclast; rheumatoid arthritis.

Figure 1

IgA and IgG autoantibody levels in synovial fluid of RA patients (A) left panel; ACPA, anti-CarP and RF IgA levels and right panel; ACPA and anti-CarP IgG levels in synovial fluid of RA patients (expressed as OD value). Dotted lines represent blanc OD values of each independent ELISA. (B) Synovial fluid profile of ACPA, anti-CarP and RF IgA and IgG levels of 26 RA patients [# IgG levels not determined]. AU, arbitrary units.

Figure 2

Cytokine release after synovial fluid immune complex stimulation correlates with IgA autoantibody levels in synovial fluid. (A) NET release by neutrophils stimulated with immune complexes isolated from healthy control (HC) serum, rheumatoid arthritis (RA) serum or synovial fluid of RA patients (RA SF). Dotted line represents unstimulated neutrophils. (B, C) Monocyte (B) IL-6 and (C) IL-8 release (pooled data from 2 independent experiments) after stimulation with immune complexes isolated from RA SF. Dotted lines represent unstimulated monocytes. (D) Correlation between immune complex-induced monocyte IL-6 release with ACPA IgA levels. (E) Correlations between monocyte IL-8 release induced by immune complexes isolated from RA SF with (left) ACPA IgA levels, (middle) ACPA IgG levels or (right) RF IgA levels Average cytokine release is displayed (n=2). Data was analyzed using two way ANOVA with Bonferroni post-hoc or using the Spearman correlation coefficient; ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 3

Enhanced osteoclast formation and activity in presence of TGF-β. (A) Osteoclast TRAcP expression (purple) in presence of M-CSF and RANK-L (upper left panel) and in presence of M-CSF, RANK-L and TGF-β (lower left panel) after 7 days culture. [Blue (DAPI) = nuclei, green (WGA) = cell membrane]. (B) CD14 expression on osteoclasts in absence (upper panel) or presence of TGF-β (lower panel) after 7 days culture. (C) Number of nuclei present in CD14- (upper panel) and CD14+ (lower panel) cells after 7 days of culture in presence of M-CSF and RANK-L. (D) Expression of αVβ3 on formed osteoclasts in absence (blue (top) histogram) or presence of TGF-β (red (second) histogram) after 7 days culture. Residing CD14⁺ cells (black histogram) and non-stained cells (dotted histogram) served as controls. (E) Bone resorption by human osteoclasts after 21 days in presence of M-CSF and RANK-L (left panel) or M-CSF, RANK-L and TGF-β (right panel) (representative of n=2 experiments). (F) Quantification of bone resorption by osteoclasts in absence of TGF-β (white bar) or presence of TGF-β (grey bar) presented as area in μm² bone resorption. Data was analyzed using unpaired student’s two-tailed T test; *p ≤ 0.05.

Figure 4

Significantly enhanced bone resorption in presence of supernatant of IgA-activated monocytes. (A) Bone resorption by osteoclasts in the presence of supernatant of unstimulated monocytes (upper left panel), BSA-stimulated monocytes (upper right panel), IgG-activated monocytes (lower left panel), or IgA-activated monocytes (lower right panel). (B) Quantification of bone resorption (area in um²) induced by osteoclasts in presence of supernatant of unstimulated monocytes (white bar), BSA-activated monocytes (light grey bar), IgA-activated monocytes (dark grey bar) or IgG-activated monocytes (black bar). Data was analyzed using one way ANOVA with Tukey post-hoc; *p ≤ 0.05.

Figure 5

Osteoclasts are activated by IgA immune complexes. (A) Expression of CD16, CD32, CD64 and CD89 on CD14 negative OCs (upper panels; dark grey histograms) and remaining CD14⁺ cells (middle panels; light grey histograms) compared to no stain (lower panels; dotted black line histograms). (B) CD89 expression on CD14^-αVβ3⁺ OCs. [White (DAPI) = nuclei, blue = CD14, red = αVβ3, green = CD89]. (C, D) IL-8 (C) and IL-6 (D) release by OCs stimulated with immune complexes isolated from synovial fluid of RA patients. (E, F) IL-8 (E) and IL-6 (F) release by unstimulated OCs (white bar) and after stimulation with BSA- (light grey bar), IgA- (dark grey bar) or IgG- (black bar) coated beads. (G) Quantification of bone resorption by OCs in presence of M-CSF and RANK-L (unstim, white bar) and added cytokines IL-6 (25 ng/ml, light grey bar) or IL-8 (10 ng/ml, dark grey bar). Data was analyzed using one way ANOVA with Tukey post-hoc; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 6

Mechanism of direct and indirect IgA-mediated osteoclast activation. In the inflamed joints of RA patients’ immune complexes consisting of IgA autoantibodies are present. Infiltrated neutrophils can form neutrophil extracellular traps (NETs) when activated with IgA-containing immune complexes. Monocytes activated with IgA-containing immune complexes produce high levels of IL-6 and IL-8 and enhance the bone resorptive capacity of osteoclasts. IgA-containing immune complexes can also directly target osteoclasts which express the Fc receptor for IgA (FcαRI). After IgA activation osteoclasts secrete IL-8 and IL-6, which are osteoclastogenic cytokines known to contribute to the formation of osteoclasts. Furthermore, IL-8 contributes to the chemoattraction and infiltration of neutrophils thereby enhancing the pro-inflammatory environment in the inflamed synovium of RA patients. Osteoclast secreted IL-8 could therefore both lead to auto stimulation and attraction of neutrophils to the inflamed area. [Created with BioRender.com].