Background Circulating levels of IL-8 is related to the bone loss associated with breast cancer metastasis. Recently, we have shown that anti-citrullinated protein antibodies (ACPA) can induce osteoclastogenesis.
Objectives We aimed to investigate the role of IL-8 and its receptors in mediating osteoclastogenesis in presence or absence of ACPAs
Methods IL-8 levels was measured by ELISA in serum of risk RA patients and synovial fluid samples of RA patients. CD14 positive monocytes were used to generate osteoclasts with M-CSF and RANKL. Inhibition of IL-8 and its receptor were tested with IL-8 neutralizing antibody or small molecule CXCR1/2 anta-agonist (Reparixin and SCH-527123) on osteoclasts (OC) in the presence or absence of ACPA. The number of the OC's were counted in light microscope after TRAP staining. Cell counting kit 8 (CCK8) assay was performed to detect cytotoxicity. CXCR1 and CXCR2 expression was analyzed using flow cytometry and immunohistochemistry during different days of osteoclasts maturation.
Results Increased IL-8 levels were observed in the serum of ACPA positive at risk RA healthy individuals and in synovial fluid of established ACPA positive RA patients. Exogenous IL-8 dose dependently increased OC numbers in presence of RANKL. IL-8 neutralizing antibody inhibited the OC numbers even in the presence of ACPA. Reparixin and SCH-527123 significantly inhibited RANKL mediated osteoclastogenesis at 100 μM concentration. No cytotoxicity of the drugs were detected with CCK8 assay. Immunohistochemistry staining confirmed the CXCR1/2 expression on OC precursors. We found high expression of IL-8 using immunohistochemistry on the inflammed RA synovium.We observed CXCR1 and CXCR2 expression with flow cytometry in the OC precursors at day 0 and in presence of RANKL the expression was completely lost at day 1 and it reappeared at day 4.
Conclusions Our data provides insights in to the importance of IL-8 autocrine loop in RANKL and ACPA mediated OC development. IL-8 receptor blockade might be a novel way of targeting bone loss in RA.
Disclosure of Interest None declared