PMC

Presence of CXCL12 RNA and CXCR4⁺ cells in the arthritic joint. (a) Synovia of three collagen type II/complete Freund's adjuvant-immunized collagen-induced arthritic (CIA) mice and three naive mice were isolated on day 35 after immunization and total RNA was purified. Reverse-transcription was performed and cDNA was subjected to quantitative PCR and normalized to the amount of 18S RNA. (b) Joints of three other collagen type II/complete Freund's adjuvant-immunized mice were washed at day 35 with PBS/FCS 2%. Cells that were harvested from the joint were stained for the presence of CD11b using fluorescein isothiocyanate (FITC)-labeled antibodies, and for CXCR4 using phycoerythrin (PE)-labeled antibodies. (c) Control staining for CXCR4 using a PE-labeled rat IgG2b isotype control antibody. One representative experiment out of two is shown.

CXCL12 stimulates and AMD3100 inhibits osteoclast differentiation. Splenocytes of three collagen type II/complete Freund's adjuvant-immunized mice were isolated and pooled. (a,b) Splenocytes were cultured for 6 days in the cups of a chamberslide, in the presence of the indicated stimuli (macrophage colony-stimulating factor (M-CSF), 20 ng/ml; receptor activator of NF-κB ligand (RANKL), 100 ng/ml; CXCL12, 0.1 μg/ml in (a), 0.5 μg/ml in (b); AMD3100, 25 μg/ml). After stimulation, cells were fixed and stained for the presence of tartrate-resistant acid phosphatase (TRAP). TRAP⁺ multinucleated (three or more nuclei) cells were counted within each cup. Bars represent averages ± standard error of the mean for four cultures. The asterisk represents p < 0.05 compared with the hatched bar (Mann-Whitney U-test). Representative pictures for TRAP-stained cultures stimulated with (c) M-CSF and RANKL alone and with added (d) CXCL12 (0.5 μg/ml) or (e) CXCL12 and AMD3100.

IL-6 levels in serum and in CXCL12-stimulated splenocyte cultures. (a) Eight mice were immunized with collagen type II/complete Freund's adjuvant (CIA) and implanted with osmotic minipumps delivering AMD3100 (four mice) at a constant rate of 600 μg/day or containing PBS (four mice). Blood was collected at day 10 of the treatment. Sera were pooled in each group and analysed for the presence of IL-6 (using a SearchLight Proteome array). Bars represent the average ± standard error of the mean of two independent experiments. (b) Splenocytes of three collagen type II/complete Freund's adjuvant-immunized mice were pooled and cultured in the absence of CXCL12, in the presence of CXCL12 (0.5 μg/ml) or in the presence of CXCL12 and AMD3100 (25 μg/ml). Supernatant was analysed after 48 h for the presence of IL-6. Bars represent the average ± standard error of the mean of three independent experiments.

CXCL12 increases and AMD3100 inhibits osteoclast activity. Splenocytes of three collagen type II/complete Freund's adjuvant-immunized mice were isolated and pooled. Cell suspensions were cultured for 6 days on a quartz substrate coated with a calcium phosphate film in the presence of macrophage colony-stimulating factor (M-CSF, 20 ng/ml) and receptor activator of NF-κB ligand (RANKL, 100 ng/ml). At day 6 media were removed, cultures were provided with fresh media and stimulated as indicated (M-CSF, 20 ng/ml; CXCL12, 0.5 ng/ml; AMD3100, 25 μg/ml). Cells were removed from the quartz substrate after 2 days and resorption pits were visualized by light microscopy. The resorbed area was measured by a bioquant image analysis system. Bars represent the mean area resorbed by 1 osteoclast (average ± standard error of the mean), measured as the area of 1 resorption pit. The asterisk represents p < 0.001 compared with the M-CSF condition (Mann-Whitney U-test). Representative pictures of resorption pits are shown for the condition stimulated with (b) M-CSF, (c) M-CSF + CXCL12 and (d) M-CSF + CXCL12 + AMD3100. The data in this figure are representative for two independent experiments.

AMD3100 in wild-type mice does not interfere with the humoral or the cellular response. At the end of the two week treatment (day 41), blood was collected from five mice out of each group. (a) Sera of individual mice were analyzed for total anti-CII IgG, using an absolute standard. Bars represent averages plus standard error of the mean of five mice. (b) Equal quantities of the sera were pooled for detection of anti-CII IgG2b and IgG1, using a standard in arbitrary U/ml; standard = 1,000 U/ml. (c) Delayed type hypersensitivity reactivity against CII. Ten mice were immunized with CII/complete Freund's adjuvant and implanted with osmotic pumps containing AMD3100 or PBS on day 7. On day 17 after immunization, five mice in each group were challenged with 10 μg of CII in the right ear and vehicle in the left. Delayed type hypersensitivity responses were measured as the percentage of ear swelling (i.e. 100 × the difference between the increase of thickness of the right and the left ear, divided by the thickness of the ear before challenge) at the indicated times. Bars represent averages ± standard error of the mean for five mice.

AMD3100 blocks CXCL12-elicited chemotaxis in vivo and in vitro. (a) Sixteen mice were immunized with collagen type II (CII) in complete Freund's adjuvant on day 0 and treated with AMD3100 or PBS in a similar way as described in the legend of Fig. 1. In vivo treatment is indicated along the X-axis. On the last day of treatment, a chemotactic assay was performed as described in Materials and methods. On that day, mice were injected with 2 μg of CXCL12 in 1 ml 0.9% NaCl (+) or 0.9% NaCl only (-) in a subcutaneous air pouch. Two hours after chemokine challenge, cells were washed out of the air pouch with 2 ml of PBS/FCS 2% and counted. Counts of the individual mice are shown (circles) and average ± standard error of the mean are indicated for each group (diamonds). (b,c) Dose-dependent inhibition by AMD3100 of CXCL12-elicited chemotaxis on total splenocytes. On day 21 post immunization with CII in complete Freund's adjuvant, spleens of three mice were pooled and a splenocyte suspension was prepared. Cell samples were pre-incubated for 10 minutes with AMD3100 at the indicated concentrations. Then, 5-μm filter inserts were loaded with 10⁶ cells and transferred to a 24-well plate containing 100 ng/ml human CXCL12 in 600μl of buffer per well. After 3.5 h of incubation, the membrane inserts were removed and the cells in the wells were collected and counted by flow cytometry. The numbers of migrated cells of one representative experiment are shown in (b). (c) The experiment was confirmed by three additional experiments and the data of the experiments were pooled and represented as the percentage inhibition ± standard error of the mean of CXCL12-elicited chemotaxis by the indicated concentrations of AMD3100.

Inhibition of collagen-induced arthritis in DBA/1 mice by treatment with AMD3100. Mice were immunized on day 0 with collagen type II in complete Freund's adjuvant and were observed for symptoms of arthritis. On day 27, when the first symptoms of arthritis appeared, the mice were divided into two groups in a way that a similar incidence and a similar average clinical score was reached in both groups. On this day, mice of one group were implanted with osmotic minipumps, delivering AMD3100 for two weeks at a constant rate of 600 μg/day. Mice of the other group were implanted with pumps containing PBS. The (a) cumulative incidence and (b) mean arthritic score ± standard error of the mean (SEM) for AMD3100-treated and control-treated mice are shown. Average group scores of arthritis were significantly different from day 30 onwards (p ≤ 0.05 on day 30; p ≤ 0.01 from day 31 till the end of the experiment, Mann-Whitney U test). (c) Evaluation of disease progression in mice with established arthritis at initiation of treatment with AMD3100. Circles represent percentage increase in scores of arthritis for individual mice at the end of the treatment. Data show the results of three individual experiments (explained in more detail in the legend of Table 1). (d-f) Histological analysis of the joints. On the last day of treatment, five mice out of both groups with a mean score representing the average group score, were selected and sacrificed. Paraffin sections of the fore and hind limbs were haematoxylin stained and histological examination was performed. Representative pictures are shown. (d) Joint of an AMD3100-treated mouse without clinical symptoms showing normal histological appearance. (e) Joints of arthritic AMD3100-treated mice show a weak infiltration of mono- and polymorphonuclear cells and hyperplasia of the synovium. (f) Joint section of a PBS-treated mouse, showing moderate to severe infiltration of leukocytes, hyperplasia and bone destruction.