Background/Purpose:

The destruction of bone is a common feature of diseases like rheumatoid arthritis (RA) and multiple myeloma (MM).  CCL3 is significantly elevated in the serum of patients with RA and MM; it is thought to contribute to leukocyte migration.  A direct link between CCL3 and the development of osteoclast-dependent erosive bone pathology has not been evaluated previously. We studied CCL3’s role in osteoclast (OC) differentiation from human macrophage precursor cells and its impact on bone resorption in vitro and in vivo.

Methods:

Human OC precursor cells (CD14^+ve monocytes) from peripheral blood were seeded onto ivory disks and differentiated into OC using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for 14 days alone or in combination with anti-CCL3 (8ng/mL) or osteoprotegerin (OPG; 8ng/mL).  CCL3 was measured in culture supernatants by ELISA over the time-course. OC formation and resorption pits were visualised by tartrate-resistant acid phosphatase (TRAP) and toluidine blue staining respectively. Murine collagen-induced arthritis (CIA) was initiated after two intradermal immunizations of complete Freund’s adjuvant and type (II) collagen (day 0 and day 21). Mice received intra-peritoneal injections (5mg/kg) of isotype control antibody (n=6) or anti-CCL3 (n=6) on day 21, 23, 25, 27, 28.  Arthritis progression was measured daily to give a clinical score. Mice were culled on day 29. Bone erosion was observed in fixed forepaws and hind limbs by x-ray (12 joints per limb) and counted.  OC counts, CCL3 concentration, percentage of the disk area resorbed, and number of eroded joints per mouse are reported as mean ± SEM. Statistical significance determined by 2-way ANOVA, Spearman’s rank co-efficient or student’s t-test as appropriate.

Results:

OC numbers per disk increased significantly over time (307 ± 81; p≤0.01 on day 14) with M-CSF and RANKL.  A concomitant 5-fold increase in CCL3 levels (day 0 to 14) was observed (p≤0.05), which significantly correlated with OC number (p≤0.01, R= 0.32).  Neutralisation of CCL3 led to a significant 3-fold reduction in OC differentiation (113 ± 41 OC/disk; p≤0.05) and were comparable to cultures supplemented with OPG.  Anti-CCL3 treatment also significantly reduced the disk area resorbed (0.49 ± 0.13%; p≤0.05) compared to controls (1.54 ± 0.4%). OPG was more potent than anti-CCL3 in this regard (disk area resorbed = 0.04 ± 0.02%, p≤0.05 versus control). In CIA, anti-CCL3 had no significant effect on joint swelling, however, radiological progression of arthritis was substantially abated. The number of erosions in anti-CCL3 treated mice was halved (7 ± 2 (p≤0.05)) versus 15 ± 2 for isotype controls.

Conclusion:

For the first time we show the potent regulatory role of CCL3 in OC differentiation and OC-directed bone resorption in both in vitro and in vivo models.  Our data unmasks the important function of CCL3 in the inflammatory milieu of a diseased joint and identifies this chemokine as target for further investigation as a marker for destructive bone disease associated with arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-macrophage-inflammatory-protein-1-alpha-ccl3-significantly-reduced-bone-resorption-in-vitro-and-the-development-of-erosive-joint-pathology-in-collagen-induced-arthritis/