title: CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production.
abstract: BACKGROUND
  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 microg/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) alpha, IL-1beta, transforming growth factor (TGF) beta) 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 microg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNFalpha, IL-1beta and TGFbeta). 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, TNFalpha, IL1-beta and TGFbeta. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA.
authors.affiliation: Research Laboratories and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Viale Benedetto XV, 16132 Genova, Italy. mcutolo@unige.it
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cutolo
authors.forename: Stefano
authors.initials: S
authors.lastname: Soldano
authors.forename: Paola
authors.initials: P
authors.lastname: Montagna
authors.forename: Alberto
authors.initials: A
authors.lastname: Sulli
authors.forename: Bruno
authors.initials: B
authors.lastname: Seriolo
authors.forename: Barbara
authors.initials: B
authors.lastname: Villaggio
authors.forename: Pierfranco
authors.initials: P
authors.lastname: Triolo
authors.forename: Paolo
authors.initials: P
authors.lastname: Clerico
authors.forename: Lamberto
authors.initials: L
authors.lastname: Felli
authors.forename: Renata
authors.initials: R
authors.lastname: Brizzolara
mesh_terms: D000069594:Abatacept; D018501:Antirheumatic Agents / Q000494:pharmacology*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000276:immunology*; D051940:B7-2 Antigen / Q000096:biosynthesis / Q000187:drug effects / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000096:biosynthesis / Q000187:drug effects* / Q000276:immunology; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D018796:Immunoconjugates / Q000494:pharmacology*; D007150:Immunohistochemistry; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D018501:Antirheumatic Agents; D051940:B7-2 Antigen; D016207:Cytokines; D018796:Immunoconjugates; D000069594:Abatacept