Background/Purpose: Increased circulating levels of Interleukin-8 (IL-8) have been linked to the bone loss associated with breast cancer metastasis. Recently, we have shown that anti-citrullinated protein antibodies (ACPAs) can induce bone loss and pain-like behavior in mice through an IL-8 dependent mechanism. We aimed to further investigate the role of IL-8 and the IL-8 receptor molecules CXCR1 and CXCR2 in osteoclasts (OC) development in the presence or absence of ACPAs.

Methods: ELISA was used to measure IL-8 in the serum of ACPA-positive patients with arthralgia and seronegative healthy controls and in the synovial fluid of RA and spondylarthropaty patients. 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 measured during OCs maturation using ELISA, RT-PCR, flow cytometry and immunostaining. 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, SCH-527123, SB-332235), in the presence or absence of ACPAs. OC numbers were counted using light microscope after TRAP staining. Cytotoxicity was monitored with Cell counting kit 8 (CCK8).

Results: Serum IL-8 levels were significantly higher in the serum of ACPA-positive patients with arthralgia, as compared to ACPA-negative healthy individuals. Synovial fluid of ACPA-positive RA patients contained significantly higher levels of IL-8 as compared to spondyloarthritis patients. Endogenous IL-8 increased gradually in the maturing OC supernatants and exogenous IL-8 dose dependently increased RANKL-mediated OC development. IL-8 neutralizing antibodies inhibited OC differentiation with or without ACPAs. Reparixin and SCH-527123 significantly inhibited the RANKL-mediated osteoclastogenesis at doses as high as 80µM concentration, without inducing detectable toxicity. SB-332235, an inhibitor characterized by higher selectivity towards CXCR2, inhibited OC development at a concentration of 10µM. CXCR1 and CXCR2 expressions were detectable on the cell membrane of macrophages and decreased during OC differentiation. Expression of the CXCR1 and CXCR2 genes in developing OCs was weak and variable. Immunohistochemistry stainings confirmed the cell surface expression of CXCR1, and to a lesser extent of CXCR2, with high amounts of both of these receptors being present intracellularly during OC maturation.

Conclusion: Serum IL-8 is a potential biomarker for ACPA-positive arthralgia and possibly for the bone loss described in these patients. Small molecule CXCR2 antagonists might provide novel therapeutic tools for targeting OCs in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-il-8-and-cxcr12-abrogates-rankl-and-acpa-mediated-osteoclastogenesis/