Session Type: Abstract Submissions (ACR)
Background/Purpose: The transcription factor NF-kB is an intracellular signaling essential for the expression of a variety of immune-response genes, including those related to pro-inflammatory cytokines . Previous studies showed that a significant downregulation of TNFα, IL-1β and IL-6 was evident for cultured human macrophages treated with CTLA4-Ig [2,3]. Therefore, we investigated the involvement of the NF-kB pathway as possible intracellular signaling target during the CTLA4-Ig modulation of cytokine production in cultured human macrophages.
Methods: Human THP1 cell line cells, differentiated by phorbol myristate acetate in macrophages, were cultured with CTLA4-Ig (100 and 500 μg/ml; 3,12, 24 hours) or without CTLA4-Ig (cnt). CTLA4-Ig/CD86 binding was evaluated by fluorescence activated cell sorter (FACS) analysis. Quantitative RT-PCR analysis (qRT-PCR) of mRNA for NF-kB, its inhibitor IKBα and for TNFα, IL-1β, IL-6 was performed after 3 and 12 hours from CTLA4-Ig treatment. Western blot (WB) analysis for NF-kB and IKBα proteins was performed after 24 hours of CTLA4-Ig treatment.
Results: FACS analysis showed a reduction of B7.2 positivity on macrophages CTLA4-Ig-treated vs. cnt, due to CTLA4-Ig/B7.2 binding. The qRT-PCR analysis of NF-kB, at 3 and 12 hours from CTLA4-Ig [100, 500 μg/ml] treatment, showed a significant downregulation (both p<0.001) vs. cnt. On the contrary, IKBα showed, at 12 hours from CTLA4-Ig [500 μg/ml] treatment a significant increase (p<0.01), while after 3 hours from [100 and 500 μg/ml] CTLA4-Ig treatment vs. cnt, resulted unchanged. The qRT-PCR analysis of inflammatory cytokines, after 3 hours from treatment, showed for CTLA4-Ig [100 μg/ml] a significant decrease of TNFα and IL-6 (p<0.05), vs. cnt. CTLA4-Ig [500 μg/ml] reduced TNFα vs. cnt in a larger extent (p<0.001). After 12 hours from CTLA4-Ig [100, 500 μg/ml] treatment, it was still evident at qRT-PCR a significant downregulation (p<0.001) for all cytokine gene expression, vs. cnt. Finally, WB analysis showed that CTLA4-Ig treatment at higher concentration [500 μg/ml] was able to reduce NF-kB protein expression and to increase IKBα expression, vs. cnt.
Conclusion: CTLA4-Ig, while it reduces the inflammatory cytokine gene expression in human macrophages, seems to promote a downregulation of the intracellular signaling linked to the NF-kB pathway, including an increased expression of its cytoplasmatic inhibitor IKBα both at gene and protein level.
References: 1. Beinke S et al. Biochem J. 2004;382:393-409; 2. Cutolo M et al. Arthritis Res Ther. 2009;11(6):R176. 3. Wenink MH et al. Ann Rheum Dis. 2012; 71(1):80-3.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-for-nf-kb-intracellular-signaling-involvement-following-ctla4-ig-abatacept-treatment-of-human-macrophages/