Background/Purpose: CTLA-4 is a surface protein on T lymphocytes, which negatively regulates the co-stimulation process between antigen-presenting cells and T cells. CTLA-4 binds to monocytes via CD80/CD86, which are precursor of osteoclast. In addition, CTLA-4 has been shown to directly inhibit osteoclast formation. However, little is known about the effect of CTLA-4 on osteoclast generation from human monocytes and monocytes exposed to inflammatory cytokines. The aim of this study was to evaluate the effect of a CTLA4-Ig (Abatacept) on osteoclastogenesis in human peripheral monocytes and TNFα-treated inflammatory monocytes.

Methods: Peripheral blood mononuclear cells are separated by Ficoll-Paque gradient centrifugation. Highly purified monocytes are prepared from prepared mononuclear cells using MACS microbeads (Pan Monocyte Isolation kit). In this study, more than 92 % of obtained cells are CD14-positive, as determined by flow cytometric analysis. Cultures were maintained in α-minimal essential medium for 7-14 days with both or either of M-CSF, RANKL and/or TNFα in the presence or absence of CTLA4-Ig. Osteoclasts were identified by TRAP staining and bone resorptive activity using the osteo assay surface multiwell plate. The expressions of CD80/CD86 on resting and TNFα-pretreated monocyte were determined by flow cytometric analysis.

Results: Peripheral monocytes from healthy donors were incubated with M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence of increasing doses of CTLA4-Ig for 5~7 days. After the culture, generated osteoclasts were determined by TRAP staining and bone resorptive activity. CTLA-4 inhibited the osteoclast generation in a dose-dependent manner (in the range of 0 to 500 μg/ml). To examine the effect of CTLA4-Ig on monocytes exposed to inflammatory cytokines, peripheral monocytes were preincubated with TNFα for 48 hours, and then cultured with RANKL. When the expression of CD80/CD86 antigens on peripheral monocytes was analysed, CD86 was only expressed on resting monocytes. Interestingly, TNFα pretreatment potently induced the expression of CD80, suggesting modification of action of CTLA-4. CTLA4-Ig more potently inhibited osteoclast generation from TNFα-treated monocytes than those from resting monocytes.

Conclusion: CTLA4-Ig directly inhibits osteoclast generation from human peripheral monocytes. In addition, the inhibitory effect of CTLA4-Ig is more potent in TNFα-treated inflammatory monocytes than resting monocytes, suggesting that CTLA4-Ig could inhibit osteoclast generation in inflammatory monocytes at inflamed joints of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ctla4-ig-directly-inhibits-osteoclast-generation-from-human-peripheral-monocytes-and-tumor-necrosis-factor-%ce%b1-treated-inflammatory-monocytes/