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AB0096 NF-KB involvement for CTLA4-IG intracellular signaling in human macrophages
  1. M. Cutolo1,
  2. P. Montagna1,
  3. S. Soldano1,
  4. A. Sulli1,
  5. B. Seriolo1,
  6. B. Villaggio2,
  7. P.F. Triolo3,
  8. L. Felli4,
  9. R. Brizzolara1
  1. 1Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine
  2. 2Clinical Academic Unit of Nephrology, Department of Internal Medicine, University of Genova, Genova
  3. 3Rheumatoid Arthritis Unit, Orthopedic Surgery Department, CTO Hospital, Turin
  4. 4Orthopedic Department, University of Genova, Genova, Italy

Abstract

Background Transcription factor NF-kB (nuclear factor kB) play an important role in the regulation of immune and inflammatory responses [1]. Previous studies showed that a significant downregulation of pro-inflammatory cytokine expression (TNFα, IL-1β and IL-6) was evident for cultured human macrophages treated with CTLA4-Ig fusion protein [2].

Objectives The aim of the study was to investigate the NF-kB pathway as possible intracellular signaling target involved in the CTLA4-Ig modulation of cytokine production (TNFα, IL-1β and IL-6) in cultured human macrophages.

Methods THP1 cell line, differentiated by PMA (0.5 μg/ml for 3 hours) into adherent macrophages, were seeded in tissue culture dishes in culture medium with CTLA4-Ig [100 and 500 μg/ml for 3 and 12 hours] or without CTLA4-Ig, for mRNA extraction and qRT-PCR analysis. At the end of incubation adherent cells were harvested and mRNA was extracted and processed by qRT-PCR analysis for NF-kB1 (p50 subunit) and for TNFα, IL-1β, IL-6. Experiments were done in triplicate.

Results The qRT-PCR analysis of NFKB1 mRNA, after 3 and 12 hours from CTLA4-Ig treatment, showed a significant downregulation (p<0.001) of the gene expression vs. cnt at both concentrations [100 and 500 μg/ml]. At the same time, the qRT-PCR analysis of inflammatory cytokine mRNA, already after 3 hours from treatment, showed that CTLA4-Ig [100 μg/ml] induced a significant decrease for TNFα and IL-6 gene expression (p<0.05), compared to untreated macrophages (cnt). CTLA4-Ig [500 μg/ml] was able to reduce TNFα gene expression vs. cnt in a larger extent (p<0.001). After 12 hours from treatment with CTLA4-Ig [100, 500 μg/ml], it was still evident a significant downregulation in cytokine gene expression for IL-1β and IL-6 vs. cnt (p<0.001). Interestingly, TNFα downregulation was not still significant after 12 hours from treatment.

Conclusions The action of CTLA4-Ig may be exerted at level of the intracellular signaling (trascription factors) by downregulating NF-kB1 gene expression, together with a significant reduction in gene expression for tested inflammatory cytokines.

  1. Beinke S et al. Biochem J. 2004;382:393-409; 2. Cutolo M et al. Arthritis Res Ther. 2009;11(6):R176.

Disclosure of Interest None Declared

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