Session Type: Abstract Submissions (ACR)
Interleukin-6 (IL-6) and the inflammatory CC-chemokine CCL3 are highly expressed in rheumatoid arthritis, juvenile idiopathic arthritis and multiple myeloma. While these mediators contribute to bone pain, osteoclast (OC) formation and bone resorption, the relationship of their activities to disease outcome remains unknown. For the first time, we measured the autocrine and paracrine regulation of CCL3 production by OC cultures in response to IL-6 (in combination with the soluble IL-6 receptor; termed IL-6 trans-signalling). Experiments identified a novel disease-activity determinant for inflammatory arthritis patients.
Human CD14+ve monocytes isolated from peripheral blood were grown on ivory disks in medium containing macrophage colony stimulating factor (10 ng/mL; m-CSF) for 7 days. Cells were subsequently differentiated into OC in medium supplemented with m-CSF (5 ng/mL) and receptor activator of nuclear factor kappa-B ligand (5 ng/mL) for 17 days. Conditioned media was recovered on day 0, 3, 7, 10, 14 and 17, and levels of CCL3, IL-6 and sIL-6R were quantified with ELISA. OC formation was visualised by staining with tartrate-resistant acid phosphatase (TRAP) on ivory disks harvested at baseline (day 0), and day 7, 10, 14 and 17 of culture. Add-back studies using anti-CCL3 antibody (0-8 ng/mL) or osteoprotegerin (OPG; 100 ng/mL) were also performed. IL-6 trans-signalling was modulated using an engineered chimeric IL-6-sIL-6R fusion protein (HDS agonist; 10 ng/mL) and soluble gp130 (sgp130 antagonist; 500 ng/mL). Cytokine release was reported as mean±SEM and statistical significance through 2-way ANOVA, Spearman’s rank co-efficient or student’s t-test as appropriate.
No OC or TRAP+ve cells were observed in culture on day 0. OC numbers reached maximum levels after 14 days in culture (338±117 per disk; P<0.001). OPG supplementation potently inhibited OC formation on day 14 (8±3 per disk, P<0.01). A significant correlation between CCL3 levels and OC number was observed (P<0.01, R= 0.32). IL-6 (140±36 pg/mL and 48±28 pg/mL) and soluble IL-6 receptor (30±9 pg/mL and 19±7 pg/mL) levels were maintained throughout the culture period (day 0 and 14 reported). In add-back experiments using a neutralizing CCL3 antibody, OC numbers decreased in a concentration-dependent manner. A maximum 2.4-fold reduction in OC was noted on day 14 (anti-CCL3; 8 ng/mL). Inhibition of IL-6 trans-signalling with sgp130 caused a 3.6-fold reduction in OC number (P<0.01). Autocrine inhibition of OC differentiation by sgp130 (142±40 pg/mL) halved CCL3 production versus controls (280±86 pg/mL). OC numbers and CCL3 were comparable in control cultures to those exposed to paracrine activation of IL-6 trans-signalling by HDS for 14 days.
For the first time, we demonstrate the importance of autocrine versus paracrine regulation of IL-6 trans-signalling in controlling OC formation in humans and unmask a hitherto unresolved mechanism involving CCL3. These data identify soluble gp130 as a potentially important disease-modifying therapy, targeting bone destruction for inflammatory arthritis patients.
L. A. Jordan,
F. L. Collins,
S. A. Jones,
E. H. Choy,
A. K. Harvey,
A. S. Williams,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibiting-autocrine-interleukin-6-il-6-trans-signalling-in-human-cd14ve-monocultures-reduces-osteoclast-differentiation/