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

Maurizio Cutolo,Paolo Clerico,Alberto Sulli,Lamberto Felli,Stefano Soldano,Renata Brizzolara,Bruno Seriolo,Pierfranco Triolo,Paola Montagna,Barbara Villaggio

doi:10.1186/ar2865

Abstract

IntroductionCo-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.MethodsAll 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.ResultsMacrophages 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.ConclusionsOptimal 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.

Highlights

Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response
Macrophages would appear to be a sensitive target for CTLA4-Ig treatment in rheumatoid arthritis (RA)
T cell response is triggered by an initial signal delivered through the T cell receptor (TCR), and it recognizes the antigenic peptide within the context of the major histocompatibility complex (MHC) molecule on the antigen-presenting cells (APC)

Summary

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). APC: antigen-presenting cells; CTLA-4: cytotoxic T lymphocyte-associated antigen-4; DMARDs: disease-modifying antirheumatic drugs; DPBS: Dulbecco's phosphate buffered saline; ELISA: enzyme-linked immunosorbent assay; IgG1: immunoglobulin G1; IL: interleukin; LPS: lipopolysaccharide; MHC: major histocompatibility complex; PMA: phorbol myristate acetate; RA: rheumatoid arthritis; RT-PCR: reverse transcriptase-polymerase chain reaction; SM: synovial macrophages; TCR: T cell receptor; TGF: transforming growth factor; TNF: tumor necrosis factor. In order to be fully activated, it needs to be followed by another signal, which is provided by the signals of the co-stimulatory molecules that are expressed on APC (such as dendritic cells, B-lymphocytes and macrophages) [9]. The activated T cells express the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) molecule, which binds the B7 molecules on APC with a 10- to 20fold greater affinity compared with CD28, and downregulates the T cell activation [12,13,14]

Methods

Results

Conclusion