Background We have shown previously in various experimental models that anti-citrullinated protein antibodies (ACPAs) can contribute to bone erosion and arthralgia through IL-8 dependent mechanisms.
Objectives In cell cultures osteoclasts (OCs) played a prominent role in the ACPA-induced IL-8 secretion and therefore we decided to characterize in detail the autocrine regulation of OC differentiation by IL-8 in the presence or absence of ACPAs.
Methods Peripheral blood CD14-positive monocytes were used to generate OCs in the presence of M-CSF and RANKL. Expression levels of IL-8 and CXCR1/2 were monitored during OCs maturation using intracellular flow cytometry, cytometric bead array, immunohistochemistry and RT-PCR. Inhibition of IL-8 and its receptors were performed in OC cultures using IL-8 neutralizing antibodies and small molecule CXCR1 and CXCR2 antagonists (Reparixin, SB-332235), in the presence or absence of polyclonal ACPAs. OC numbers were counted using light microscope after TRAP staining.
Results Macrophages and developing OCs secreted high levels of IL-8 in response to the cytokine M-CSF. IL-8 expression kinetic was characterized by transient peaks when new cytokine was added, followed by very low level of steady-state production. In the presence of ACPAs IL-8 production increased 3–4 fold during late phases of OC differentiation. With the help of IL-8 neutralizing antibodies or inhibitors acting on the IL-8 binding chemokine receptors CXCR1 and CXCR2 we showed that the M-CSF induced IL-8 plays a crucial role in OC differentiation in the presence of exogenous RANK-L. In the presence of ACPAs OC differentiation was further enhanced in response to the ACPA-mediated increase in IL-8 production. The CXCR1 and CXCR2 inhibitors Reparixin and SB-332235 significantly inhibited the RANKL-mediated osteoclastogenesis at 100μM and 10μM respectively, both in the presence or absence of ACPAs. By using the inhibitors at low concentrations we could counteract the ACPA-mediated increase in OC development without interfering with steady state OC differentiation.
Conclusions Our findings further support an important role for IL-8 in bone biology and in the ACPA-mediated OC differentiation both under physiological conditions and in the presence of ACPAs. Together with previous data showing in vivo, our data indicated that small molecule CXCR1 and CXCR2 antagonists could provide novel therapeutic tools for targeting OCs in ACPA+ individuals.
Disclosure of Interest None declared