CIA is a useful model of RA for studying inflammation, autoimmunity and arthritis. Antibodies specific for collagen play a major role in the induction of the disease, and immune complexes formed by the autoantibodies contribute to inflammation and tissue destruction1
However, the mechanism of immune complex-mediated modulation of CIA is not fully understood. Our aim was to investigate the impact of FcγRII/III targeted complexes composed of a collagen epitope peptide and 2.4G2 scFv on the induction and maintenance of CIA.
The CII-peptide was originally described as a triple helical conformational epitope of collagen spanning the 359–369 peptide region and was reported to be target for collagen specific antibodies of mice and for the sera of some RA patients.22
We hypothesized that the memory B-cells of collagen primed mice would recognize FcγRII targeted CII-peptide, which in turn might switch off antibody production through the FcγRIIb dependent B-cell inhibition, thus ameliorating disease symptoms. Supplementary file 2
shows that indeed, 2.4G2 scFv is able to inhibit intracellular Ca2+
mobilization in B-cells when it is co-crosslinked with BCR. However, we could not detect the binding of either 2.4G2 scFv or its complexes with CII-peptide to germinal center B-cells in spleen sections when they were applied in vivo.
Comparing CIA scores in the four experimental groups (suboptimally collagen immunized mice treated with 2.4G2 scFv-CII-peptide complexes, CII-peptide tetramers or 2.4G2 scFv tetramers), surprisingly we found that all molecular constructs elevated arthritic scores, thus significantly aggravating disease activity. By comparison, a mixture of monomeric CII-peptide and monomeric scFv did not elevate the arthritic scores as compared to controls. None of the constructs induced any sign of disease when administered into DBA/1 mice that were not immunized previously with collagen (data not shown). We assumed that the elevation of arthritic scores of collagen primed mice by FcγRII/III targeted CII-peptide and 2.4G2 scFv tetramers might be the result of targeting FcγRII/III positive cells,24
including dendritic cells (DC) and macrophages.
To identify the type of cells that are able to bind the various complexes, we monitored their in vitro and in vivo binding to spleen cell subsets of collagen immunized DBA/1 mice. In vitro assays showed that approximately half of the spleen B-cells from collagen-immunized mice bound 2.4G2 scFv-containing complexes or tetramers. This finding is in line with an earlier observation demonstrating that FcγRIIb expression is markedly downregulated in germinal center B-cells.40
The in situ analysis of the in vivo binding of intravenously injected 2.4G2 scFv-CII-peptide complexes showed that these preferentially bound to MARCO positive marginal zone macrophages, while a weaker reactivity of both the complexes and the peptide tetramers could also be observed with CR1/CR2high
cells. The T-zone location of these cells precludes their identification as follicular dendritic cells, but their morphology indicates some relatedness to DC-lineage. Flow cytometric analysis showed that about 40% of dendritic cells and more than half of the F4/80-positive macrophages bound 2.4G2 scFv-containing constructs ex vivo. Since the CII-peptide tetramers, besides binding to a small proportion of B-cells, also bound to macrophages and dendritic cells, we suppose that peptide-specific IgG in the serum of collagen primed mice mediates this binding (). It was reported earlier that antigen complexed with specific IgG can be taken up by DCs in a nondegradative pathway via FcγRIIb and subsequently re-expressed at the DC surface thus promoting B cell proliferation and IgG2a antibody production.41
Thus we assumed that 2.4G2 scFv-CII-peptide complexes binding to FcγRIIb on DC promote the peptide specific IgG2a synthesis in the complex-treated mice.
Figure 8 Possible ways to regulate CIA by complexes targeted to FcγR. (A) On dendritic cells: mixed complexes of 2.4G2 scFv and CII-peptide or tetramers of CII-peptides may directly or indirectly, via peptide-specific IgG, bind to FcγRII/III, thus (more ...)
To find out if CII-peptide-containing complexes and tetramers induce antibody synthesis, we compared the collagen-specific and the CII-peptide-specific IgG titers in the four experimental groups. Although administration of the various constructs into collagen-pre-immunized mice did not significantly modify the collagen-specific IgG titer, the FcγRII/III targeted CII-peptide complexes significantly elevated the peptide-specific IgG titer as compared to the nontreated and tetramer treated groups. These data indicate that although CII-peptide specific IgG is present in the sera of all groups of collagen pre-immunized mice, FcγRII/III targeted CII-peptide is significantly more efficient in eliciting peptide-specific antibody responses as compared to the CII-peptide alone. Serological data also demonstrated that the FcγRII/III targeted CII-peptide complexes enhanced the synthesis of the inflammatory IgG2a isotype, which is associated with a Th1 response.42
CII-peptide specific IgG2a in the serum would allow CII-peptide-IgG2a complexes to be formed in mice receiving CII-peptide containing constructs. IgG2a and IgG2b bind to stimulatory FcγRIV expressed by macrophages and neutrophils at 10-to-100-fold higher affinities compared to its inhibitory counterpart, FcγRIIb, thus predicting that the production of these IgG subclasses could less effectively be blocked by FcγRIIb-mediated suppression.43
Based on these findings, we suppose that CII-peptide–IgG2a complexes boost the inflammation in collagen primed DBA/1 mice due to their binding to FcγRIV. This is in line with a recent report showing that the murine high-affinity IgG receptor FcγRIV is sufficient for autoantibody-induced arthritis.44
The outcome of immune complex binding to FcγR positive cells depends on the balance between activating and inhibiting FcγR. It was reported earlier that macrophages derived from CIA susceptible mice show a disregulated FcγR expression resulting in a prolonged expression of the activating FcγRI and FcγRIII and down regulation of FcγRIIb.45
Thus FcγRII/III targeted CII-peptide complexes and tetramer 2.4G2 scFv may prominently bind to FcγRIII on macrophages in collagen primed DBA/1 mice, eliciting the production of proinflammatory cytokines such as IL-1 and TNFα.21
Proinflammatory cytokines are important regulators of the synovial inflammation in RA.46
Th1 and Th17 cells producing IFNγ, IL-17 and IL-23 have been shown to aggravate RA,38
moreover, IL-17 together with TNFα was found to be predictive for poor outcome in RA.47
In CIA the IL-23/IL-17 axis is critical for the development of autoimmune arthritis.36
It was also shown that IL-17-deficient mice were resistant to CIA.48
Furthermore, IFNγ and IL-17 amplified FcγR mediated cartilage destruction in murine immune complex-mediated arthritis.20
We found that IFNγ, TNFα, IL-17 and IL-10 were secreted in the in vitro culture of splenocytes of collagen preimmunized mice exposed to FcγRII/III targeted CII-peptide complexes. Further, protein microarray analysis showed a substantial alteration in the secreted cytokine and chemokine profile two hours after injection of the complexes into collagen primed mice. A considerably increased level of IL-1, IL-3, IL-6, IL-7, IL-10, IL-12, IL-17, IL-23 and TNFα was detected and the secretion of chemokines CXCL13, MIP-1, and MIP-2 was also enhanced. CII-peptide tetramers stimulated IL-10, IL-17, IL-23, and MIP-2 production, while 2.4G2 tetramers that may crosslink the activating FcγRIII, enhanced G-CSF, IL-6, IL-10, and MIP-2 secretion.
Macrophage activation is induced by – among others – the action of immune complexes on activating FcγR, triggering the production of a number of inflammatory mediators.49
Macrophage inflammatory protein 1α (MIP-1α/CCL3) is produced by RA synovial tissue-lining cells and interstitial macrophages,51
and it is induced by TNFα.49
MIP-1α is chemotactic for monocytes, T, B and NK cells, basophils and eosinophils, and abundant MIP-1α was found in RA synovial fluid.53
Expression and contribution of MIP-1α and macrophage inflammatory protein 2 (MIP-2) and also of IL-10 during the evolution of CIA has been described. IL-10 appears to be an important immunomodulator of the pathogenesis of CIA, regulating the expression of MIP-1α and MIP-2.37
IL-23 produced by antigen-stimulated dendritic cells and macrophages is one of the essential factors required for the survival and/or expansion of Th17 cells, which produce IL-17, IL-17F, IL-6, and TNFα. The IL-23/IL-17 axis also plays a key role in the development of autoimmune arthritis in humans.54
IL-23 acts on dendritic cells and macrophages in an autocrine/paracrine manner to stimulate the generation of proinflammatory cytokines, such as IL-1, IL-6, and TNF-α in autoimmune inflammatory diseases.36
A 2-year study on a cohort of RA patients has demonstrated that synovial membrane mRNA levels of IL-1beta, TNF-α, IL-17, and IL-10 were predictive of damage progression. IL-17 was synergistic with TNF-α.47
The synovial milieu in established RA contains various macrophage- and synovial-fibroblast derived cytokines, such as IL-1β, IL-6, IL-7, IL-12, IL-15, IL-18, IL-23p19, and TGFβ that can support the expansion and differentiation of Th1 and/or Th17 cells.49
Bone resorption in RA is induced by osteoclasts, and osteoclast differentiation is achieved by the actions of TNF and IL-1, as well as of IL-17, produced by Th17 cells, and IL-7, produced by synovial fibroblasts. 49
These data show that evidence suggesting rheumatoid arthritis as a primarily Th17-/IL-17-dependent autoimmune inflammatory disease is rapidly accumulating.
Taken together, our data suggest that 2.4G2 scFv-CII-peptide complexes and the in vivo formed IgG2a-CII-peptide immune complexes may bind to FcγRIII and/or to FcγRIV, inducing the secretion of IL-23 from dendritic cells and macrophages, which in turn triggers Th17 cells to secrete IL-17, IL-6 and TNFα. Additionally, the enhanced production of further inflammatory cytokines and chemokines was detected after the intravenous injection of the complexes and tetramers. Treatment of collagen preimmunized DBA/1 mice with the FcγRII/III targeted complexes also resulted in increased secretion of IL-3 and G-CSF. Recent data indicate that IL-3 produced by CD4+ T-cells in the presence of CD11b+ macrophages has an important role in the early phase of CIA by activating basophils, thus considerably expanding the range of possible effector cells.55
Furthermore, it was reported that daily injections of M-CSF or G-CSF, 20–24 days after primary immunization with type II collagen, exacerbate disease symptoms in suboptimally immunized DBA/1 mice, showing that M-CSF and G-CSF can be proinflammatory in CIA.56
Among the chemokines induced by the FcγRII/III targeted CII-peptide complexes BLC (CXCL13), MIP1 and MIP-2 are the most important. B lymphocyte chemoattractant cytokine CXCL13 controls follicle formation and may facilitate ectopic lymphoid organogenesis in the synovium and the local production of tissue-specific auto-antibody.57
CXCL13 is expressed in the synovial tissue of RA patients and its blockade reduces the severity of CIA, indicating that CXCL13 plays an important role in the development and pathogenesis of the disease.58