Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004;6(3):R220-31.
doi: 10.1186/ar1167. Epub 2004 Mar 12.

Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Bert De Klerck  1 , Isabelle Carpentier, Rik J Lories, Yvette Habraken, Jacques Piette, Geert Carmeliet, Rudi Beyaert, Alfons Billiau, Patrick Matthys
Affiliations

Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Bert De Klerck et al. Arthritis Res Ther. 2004.

Abstract

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b+ myeloblasts. Both disease manifestations are more pronounced in interferon-gamma receptor knock-out (IFN-gammaR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b+ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-gammaR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-kappaB ligand (RANKL) or with tumour necrosis factor-alpha (TNF-alpha). Significantly larger numbers of such cells could be generated from splenocytes of IFN-gammaR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-alpha-induced osteoclast formation. Splenocyte cultures of IFN-gammaR KO mice also produced more TNF-alpha and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-gammaR KO mice contained comparatively low levels of pro-interleukin-1beta (pro-IL-1beta) and pro-caspase-1, indicating more extensive conversion of pro-IL-1beta into secreted active IL-1beta. These observations provide evidence that all conditions are fulfilled for the expanding CD11b+ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-gammaR KO mice to CIA.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Accelerated collagen-induced arthritis (CIA) in interferon-γ receptor knock-out (IFN-γR KO) mice is accompanied by CD11b+ splenocyte expansion and osteoclast formation in the joints. Mice were immunised with collagen type II in complete Freund's adjuvant. (a) Accelerated disease onset and more severe arthritic scores in IFN-γR KO mice than in wild-type mice. (b) Expansion of the CD11b+ splenocyte population in IFN-γR KO mice 27 days after immunisation. Splenocytes were obtained from three mice, counted and then pooled for flow cytometric analysis; numbers indicated are per spleen. (c,d) Haematoxylin staining on paraffin sections of the joints on day 27 after immunisation, showing bone erosion and multinucleated giant cells in IFN-γR KO mice (arrows in (c) and detail in inset). (d) Section of wild-type mouse joint, showing normal histological appearance. (e) Tartrate-resistant acid phosphatase (TRAP) staining on paraffin sections of the joint of IFN-γR KO mice, showing multinucleated giant cells (arrows; detail in inset) staining positive for TRAP. TRAP+ multinucleated cells (three or more nuclei) can be considered to be osteoclasts. (f) CD11b+ cells in CIA. Staining with anti-mouse CD11b antibody demonstrating the presence of both CD11b+ (brown) mononuclear cells and multinuclear osteoclast-like cells (arrow; enlarged in inset). The section was counterstained with haematoxylin. Sections that were stained with an isotype control antibody revealed no positive staining (not shown). Bars in the pictures and insets represent, respectively, 100 μm and 10 μm.
Figure 2
Figure 2
Increased osteoclast formation from splenocytes of interferon-γ receptor knock-out (IFN-γR KO) mice. Splenocytes of IFN-γR KO (KO) mice and wild-type (WT) mice were isolated. Mice were either naive or had been immunised with collagen type II in complete Freund's adjuvant 21 days previously. Cells were stimulated for 6 days in chamber slide cups with 20 ng/ml macrophage colony-stimulating factor (M-CSF) and 100 ng/ml receptor activator of NF-κB ligand (RANKL) (a) or 20 ng/ml M-CSF and 20 ng/ml tumour necrosis factor-α (TNF-α) (b). After stimulation, cultures were fixed and stained for the presence of tartrate-resistant acid phosphatase (TRAP). (a,b) TRAP+ multinucleated (three or more nuclei) cells were counted within each cup. In each group, bars represent averages ± standard error of the mean for five mice. *P < 0.05 compared with wild–type mice (Mann-Whitney U-test). (c,d) Representative pictures of TRAP-stained cultures of RANKL-stimulated IFN-γR KO (c) and wild-type (d) splenocytes. Insets show details of multinucleated TRAP+ cells. (e,f) Osteoclast activity after stimulation by RANKL of IFN-γR KO (e) and wild-type (f) splenocyte cultures, as analysed by their ability to resorb a calcium phosphate film coated on a quartz substrate. Sites of resorption are indicated by arrows. CIA, collagen-induced arthritis.
Figure 3
Figure 3
Flow cytometric analysis for receptor activator of NF-κB (RANK) on splenocytes of immunised interferon-γ receptor knock-out (IFN-γR KO) and wild-type mice. Splenocytes were isolated on day 21 after immunisation. Cells were incubated with phycoerythrin-labelled anti-CD11b antibody, biotinylated anti-RANK antibody and fluorescein isothiocyanate-labelled streptavidin. The grey line represents staining with an irrelevant biotinylated IgG. RANK expression was analysed both within the CD11b- (left panels) and within the CD11b+ (right panels) splenocyte population. The black line represents staining with anti-RANK–biotin/streptavidin–phycoerythrin. The total numbers of RANK+ cells within the CD11b+ population of the IFN-γR KO (upper right panel) and of the wild-type (lower right panel) splenocytes are indicated, together with the percentage that they represent of the total splenocyte population (each picture is representative for one mouse out of three).
Figure 4
Figure 4
Production of receptor activator of NF-κB ligand (RANKL) and tumour necrosis factor-α (TNF-α) in vitro and expression of macrophage colony-stimulating factor (M-CSF) and RANKL in vivo. (a,b) RANKL and TNF-α concentrations measured in supernatant of anti-CD3 antibody-stimulated splenocytes. Splenocytes of naive and immunised mice (day 21 after immunisation) were cultured in the presence of M-CSF and stimulated with 1 μg/ml anti-CD3 antibody. RANKL (a) and TNF-α (b) detected in supernatants by enzyme-linked immunosorbent assay 6 days after stimulation. *P < 0.05 compared with wild-type mice (Mann–Whitney U-test). (c) Reverse transcriptase PCR performed on RNA of isolated inflamed synovia of two interferon-γ receptor knock-out (IFN-γR KO) mice (KO1, KO2) and two wild-type mice (WT1, WT2) showing transcription of RANKL and M-CSF within the inflamed synovium. The housekeeping gene β-actin was used to normalise the levels of cDNA. (d) Analysis of calcitonin receptor expression level by real-time quantitative PCR on synovium and spleen from three wild-type and three IFN-γR KO mice. Values are the numbers of calcitonin receptor mRNA copies per 1000 copies of hypoxanthine transferase. CIA, collagen-induced arthritis.
Figure 5
Figure 5
Expression of TNF receptor associated factor (TRAF)6, caspase-1 and interleukin-1β (IL-1β) in splenocytes of interferon-γ receptor knock-out (IFN-γR KO) mice (KO) and wild-type mice (WT), both naive and immunised with collagen type II in complete Freund's adjuvant (CII/CFA) (day 21 after immunisation). Splenocyte suspensions of three mice within each group were pooled and lysed. Total protein (30 μg) was loaded for electrophoresis and blotted. The blotting membrane was incubated with anti-TRAF6 or anti-TRAF2 antibody (a), anti-caspase-1 antibody (b) and anti-IL-1β antibody (c). (a) Degradation of TRAF6 in CII/CFA-immunised IFN-γR KO mice: there is unaltered expression of TRAF2, demonstrating that TRAF6 degradation is not caused by an overall higher protease activity in mutant mice. (b) Differential expression of the caspase-1 isoforms: inactive pro-caspase-1, intermediate caspase-1 form and the active 20 kDa form (p20). (c) Inactive pro-IL-1β mainly detectable only in wild-type mice. CIA, collagen-induced arthritis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Manoury-Schwartz B, Chiocchia G, Bessis N, Abehsira-Amar O, Batteux F, Muller S, Huang S, Boissier M-C, Fournier C. High susceptibility to collagen-induced arthritis in mice lacking IFN-γ receptors. J Immunol. 1997;158:5501–5506. - PubMed
    1. Vermeire K, Heremans H, Vandeputte M, Huang J, Billiau A, Matthys P. Accelerated collagen-induced arthritis in interferon-γ receptor-deficient mice. J Immunol. 1997;158:5507–5513. - PubMed
    1. Matthys P, Vermeire K, Mitera T, Heremans H, Huang J, Schols D, Dewolf-Peeters C, Billiau A. Enhanced autoimmune arthritis in IFN-γ receptor-deficient mice is conditioned by mycobacteria in Freund's adjuvant and by increased expansion of Mac-1+ myeloid cells. J Immunol. 1999;163:3503–3510. - PubMed
    1. Kronenberg HM. Developmental regulation of the growth plate. Nature. 2003;423:332–336. doi: 10.1038/nature01657. - DOI - PubMed
    1. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423:337–342. doi: 10.1038/nature01658. - DOI - PubMed

Publication types

MeSH terms