ACR Meeting Abstracts

ACR Meeting Abstracts

Abstract Number: 2164

Evidence for NF-Kb Intracellular Signaling Involvement Following CTLA4-Ig (Abatacept) Treatment of Human Macrophages

Renata Brizzolara1, Paola Montagna1, Stefano Soldano2, Alberto Sulli3, Bruno Seriolo4, Barbara Villaggio5, Pierfranco Triolo6, Lamberto Felli7 and Maurizio Cutolo8, 1Department of Internal Medicine, Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy, 2Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy, 3Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy, 4Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genoa, Italy, 5Research Laboratory of Nephrology, Department of Internal Medicine, University of Genova, Genova, Italy, 6Rheumatoid Arthritis Unit, Orthopedic Surgery Department, CTO Hospital, Turin, taly, Turin, Italy, 7Orthopedic Department, University of Genova, Genova, Italy, 8Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords:

Session Information

Title: Rheumatoid Arthritis Treatment - Small Molecules, Biologics and Gene Therapy

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 [1]. 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.

Disclosure:

R. Brizzolara,
None;

P. Montagna,
None;

S. Soldano,
None;

A. Sulli,
None;

B. Seriolo,
None;

B. Villaggio,
None;

P. Triolo,
None;

L. Felli,
None;

M. Cutolo,
None.

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