Research article

CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

Maurizio Cutolo¹ , Stefano Soldano¹ , Paola Montagna¹ , Alberto Sulli¹ , Bruno Seriolo¹ , Barbara Villaggio² , Pierfranco Triolo³ , Paolo Clerico³ , Lamberto Felli⁴ and Renata Brizzolara¹

¹  Research Laboratories and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Viale Benedetto XV, 16132 Genova, Italy

²  Clinical Academic Unit of Nephrology, Department of Internal Medicine, University of Genova, Viale Benedetto XV, 16132 Genova, Italy

³  Rheumatoid Arthritis Unit - Orthopedic Surgery Department, CTO Hospital, Via Zuretti 10126 Turin, Italy

⁴  Orthopedic Department, Largo Rosanna Benzi, University of Genova, 16132 Genova, Italy

author email corresponding author email

Arthritis Research & Therapy 2009, 11:R176doi:10.1186/ar2865

Published:	23 November 2009

Abstract

Introduction

Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.

Methods

All macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 μg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) α, IL-1β, transforming growth factor (TGF) β) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.

Results

Macrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 μg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNFα, IL-1β and TGFβ). Data were confirmed by WB and RT-PCR analysis.

Conclusions

Optimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNFα, IL1-β and TGFβ. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA.