Background
It is unknown whether and how
endogenous glucocorticoids (GC) contribute to the initiation and maintenance of rheumatoid arthritis (RA) and other inflammatory diseases [
1‐
7]. We previously studied the role of endogenous GC in the K/BxN serum-induced arthritis mouse model of RA [
8]. This model is T cell-independent as arthritis is elicited by antibodies even if the recipients are devoid of lymphocytes [
9,
10]. Immune complexes of arthritogenic anti-glucose-6-phosphate isomerase autoantibodies attract and activate neutrophils and macrophages at the cartilage surface through Fc receptor binding (particularly FcγRIII) and activation of complement factors from the initial part of the alternative complement pathway [
11‐
15]. This induces the release of pro-inflammatory cytokines including interleukin (IL)-1 and tumour necrosis factor-α [
16].
We have recently demonstrated that inactivation of endogenous glucocorticoids in osteoblasts by overexpression of the GC-inactivating enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), results in attenuation of K/BxN serum-induced arthritis [
8]. These findings indicated an immunostimulatory role of endogenous glucocorticoids and suggested that osteoblasts modulate the immune-mediated inflammatory response via a GC-dependent pathway. While K/BxN serum-induced arthritis is principally a T cell-independent model of rheumatoid arthritis, osteoblasts and osteoblastic GC-signalling may or may not modulate the T cell-mediated inflammatory response in other models of arthritis. For example, the crosstalk between T cells and osteoblasts is known to be important in intermittent parathyroid hormone induced bone formation, involving Wnt signalling by Wnt10b [
17], which we have previously shown to be GC-dependent in osteoblasts during development [
18]. Osteoblasts interact with T cells also by the production of cytokines such as IL-6 [
19], and IL-6 expression in osteoblasts is likely to be GC-regulated [
20].
In order to test whether or not the modulation of the inflammatory response by osteoblasts involves T cells, we studied the effects of disrupted osteoblastic GC-signalling in the T cell-dependent model of antigen-induced arthritis (AIA) [
21,
22]. In this model, an adaptive immune response is initiated by immunisation against the non-self antigen methylated bovine serum albumine (mBSA). Local re-injection into the knee joint induces a mainly CD4+ T cell-mediated arthritis [
21,
22].
Discussion
In sharp contrast to our previous findings in K/BxN serum-induced arthritis [
8], the present study demonstrates that murine antigen-induced arthritis (AIA) is unaffected by disruption of GC signalling in osteoblasts. The most important variables for the decision to reject the hypothesis were clinical signs and histology. Neither in acute nor in prolonged AIA did any clinical signs of arthritis differ significantly between transgenic and WT mice. The impression that the WT- and tg-mice in the short-time and long-time experiments react differently to arthritis induction (Figure
1) is due to the lower number of animals in the long-time experiments. This was confirmed via histological assessment of inflammatory activity, cartilage degradation or bone erosion, where also no significant differences were detectable in both experiments.
The immunological and inflammatory profiles of AIA differ profoundly from those of K/BxN arthritis (Table
2). In AIA, disruption of GC-signalling in osteoblasts is not relevant, probably because the antibody- mediated inflammatory response is of minor importance and the T cell response overcomes other effects. Antigen-specific T cells, in particular CD4+ T helper cells, generated within the adaptive immune response initiated through immunisation, play a major role in AIA as arthritis in this model cannot be induced in T cell-deficient mice [
21,
22,
30]. The antibody-mediated inflammatory response is present in AIA as well, but apparently this is of minor importance here. This is evidenced by the observations that (i) mBSA antibodies-containing serum can only induce very weak arthritis and (ii) antibody-producing B cells are not at all required for induction of arthritis [
21,
22,
30]. The mBSA challenge induces a significant neutrophil migration which peaks at 24 hours and subsides by seven days after challenge [
31,
32]. Fc Receptors are involved in this activation [
33,
34], but – in contrast to exclusively antibody-mediated arthritis models – complement appears to play a minor role. This conclusion has been derived from findings which demonstrate that flare-up reactions of AIA are complement-independent, and that neutrophils occur in the synovial tissue of complement-depleted mice as well [
35].
Table 2
Immunological and inflammatory profiles of AIA and K/BxN models
T cells | | |
B cells | | |
Antibodies | Antibodies unnecessary/insufficient to induce arthritis to the full extent [ 21, 22, 30] | Antibodies necessary/sufficient to induce arthritis in full extent [ 9] |
Complement | Complement-independent [ 31] | Complement-dependent [ 12] |
Fc receptors | | |
Neutrophils, macrophages | Neutrophils important [ 34, 35], but complement-independent [ 31] | Neutrophil-dependent, macrophage-dependent [ 13, 14] |
Cytokines | IL-1, TNF-α, IL-6, IL-17, RANKL important [ 22, 36] | IL-1-, TNF-α-dependent [ 16] IL-6-, osteopontin-independent [ 16, 37] |
In contrast, in antibody-mediated arthritis, such as K/BxN serum-induced arthritis, T cells are not required. Arthritis is provoked by the antibodies even if the recipients are devoid of lymphocytes [
9,
10]. Immune complexes of arthritogenic auto-antibodies act through Fc receptors and the complement network [
11,
12], activating neutrophils and macrophages which have been shown to be essential for induction of K/BxN serum-induced arthritis (whereas mast cells have recently been found to be apparently not mandatory) [
13‐
15,
38]. Therefore, our results suggest that the GC-dependent pathway by which osteoblasts modulate the inflammatory response is T cell-independent. Osteoblasts appear to impact the immune complex-mediated inflammatory response via a GC-dependent pathway.
The alternative complement pathway is essential in K/BxN serum-induced arthritis [
12]. Complement factors are mainly generated in the liver. However, osteoblasts have been shown to express complement factors [
39], and in endothelial cells, complement factor expression has been found to be GC-dependent [
40]. Hence, osteoblasts may produce alternative complement factors in a GC-dependent way (e.g. C3, factor B, factor H), and reduction of these osteoblastic alternative complement factors could be responsible for attenuation of arthritis in Col2.3-11β-HSD2-transgenic mice. Other pro-inflammatory factors in antibody-mediated arthritis – which may be generated by osteoblasts in a GC-dependent way – are urokinase-type plasminogen activator (u-PA) [
41‐
43], matrix metalloproteinases (MMP) [
18,
43] and macrophage migration inhibitory factor (MIF) [
44‐
46]. Osteopontin and IL-6 are rather unlikely to be responsible because they play no essential role in K/BxN arthritis [
16,
37].
We found no differences in multiplex analysis of serum cytokine levels between arthritic and control mice for WT mice, and only IL-1α, IL-12 p40 and IL-13 for tg mice. This is in accordance with a recently published follow up (from 7 hours to 14 days) multiplex analysis of 24 cytokines in synovial fluid and sera of rats developing antigen-induced arthritis [
47]. Cytokine concentrations in sera also showed only little variation here, whereas between cytokine concentrations in arthritic synovial fluid and histological or clinical parameters some correlations were established [
47]. We agree with the authors, that “such results are consistent with the local and monarticular nature of AIA, rendering the amount of cytokines produced within a single diseased joint prone to be reduced by degradation in the lymphatic system or by dilution into the bloodstream or both” [
47]. The relevance of the differences for IL-1α, IL-12 p40 and IL-13 between AIA WT and tg mice remains unclear. IL-12 p40 and IL-13 levels were lower in tg mice in comparison to WT mice after 14 days, but paradoxically higher after 28 days. In our study of K/BxN serum-induced arthritis, IL-12 p40 levels had not been different, and IL-6 and M-CSF levels tended to be altered in tg mice in comparison to WT mice (IL-1α and IL-13 levels were not determined) [
8]. There is indeed evidence for a (endogenous) glucocorticoid modulation of IL-1α, IL-12 p40 and IL-13 [
48‐
51]; however, such a modulation in osteoblasts apparently is of no relevance for AIA.
We did not measure a decrease of BV/TV or an increase in osteoclast covered bone surface due to AIA-arthritis in WT-mice compared to the control group of WT-mice, probably because μCT and histomorphometric measurements were done at the contralateral tibia. Even if the severity of arthritis at the arthritic knee in this model reached a normal extent, comparable to what is known in literature [
52], apparently there are no systemic effects on bone due to the monarticular nature of AIA. This is in line with the cytokine measurements (see above). In our studies of K/BxN arthritis we had seen a systemic effect of arthritis on bone in μCT and histomorphometry at the tibia in WT mice, which was prevented in tg mice [
8]. The extent of erosions achieved in the arthritic knee in AIA was low in short and long-term experiments, consistent with previous investigations [
53,
54].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CS planned and performed experiments, analysed and interpreted data, performed the histological scoring and drafted the manuscript; EW performed experiments, analysed and interpreted data, performed the histological scoring and carried out the micro-CT and histomorphometry analysis; JWT did experiments and performed the histological scoring; AL did pre-experiments; TG carried out the cytokine analysis and planned the study; DH performed the statistical analysis; HZ, MJS and FB supervised the project, planned the study and reviewed the manuscript. All authors read and approved the final manuscript.