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

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.

Abatacept modulates proinflammatory macrophage responses upon cytokine-activated T cell and Toll-like receptor ligand stimulation

“…Abatacept might directly activate certain pathways in macrophages via CD80/86, resulting in the inhibition of cytokine release upon Tck stimulation, as was suggested by Cutolo et al 12. They demonstrated, using a concanavalin-A-activated Jurkat T cell line, that abatacept was capable of reducing the (immunocytochemically) detectable levels of IL-6 and TNFα in RA synovial tissue macrophages, underscoring the clinical relevance of the results described in this paper.…”

“…Activation of mouse DCs via CD80/86 by CTLA4-expressing T cells resulted in the production of IFNγ by the DCs 11. It was recently suggested that CTLA4-Ig, via a direct effect on macrophages, reduces the ability of macrophages to produce cytokines upon stimulation with concanavalin-A-activated Jurkat T cells 12. We therefore investigated whether abatacept could influence the RA disease course by diminishing the proinflammatory cytokine production by macrophages induced upon contact with cytokine-activated T cells and TLR ligands.…”

Treatment with abatacept prevents experimental dermal fibrosis and induces regression of established inflammation-driven fibrosis

“…These data suggest that abatacept might exert alternate modes of action independent from T-cell activation blockade, as it was described in collagen-induced arthritis 26. This might also be consistent with additional effects of abatacept observed on others cell types expressing CD80/CD86 on their surfaces, including B cells27 28 and monocytes 29 30. However, we observed that abatacept failed to protect CB17-SCID mice from the development of bleomycin-induced dermal fibrosis, which strongly supports that T cells directly drive the antifibrotic properties of abatacept in this model.…”

CTLA4-Ig interferes and downregulates the proinflammatory activities of rheumatoid synovial macrophages in monoculture

“…Si può ipotizzare che il blocco dei recettori B7.1/B7.2 tramite CTLA4-Ig potrebbe altresì esercitare effetti diretti sulle APC, in particolare sui macrofagi che, come noto, giocano un ruolo fondamentale in diverse fasi della sinovite che si accompagna all'AR. Studi in vitro che abbiamo condotto in precedenza hanno dimostrato la presenza della molecola B7.2 sulla superficie di macrofagi sinoviali (MS) ottenuti da pazienti affetti da AR attiva e inoltre hanno evidenziato i possibili effetti anti-infiammatori di CTLA4-Ig in co-colture di MS AR e cellule T attivate (9). Pertanto, in questo lavoro ci proponiamo di valutare i MS come possibili cellule target per il trattamento con CTLA4-Ig, in assenza della popolazione linfocitaria T, analizzando gli effetti di CTLA4-Ig sulla produzione di citochine pro-infiammatorie quali IL-6, TNFalfa e IL-1beta da parte degli stessi MS.…”

“…sentate da MS AR sono state incubate per 24 ore in terreno addizionato con CTLA4-Ig alla concentrazione di [500 μg/ml], la concentrazione più significativa usata in base ai risultati ottenuti in precedenti studi farmacologici clinici e sperimentali (9). Il fenotipo dei macrofagi è stato confermato tramite colorazione immunocitochimica con anticorpo HAM56 (Dako, Carpinteria, CA, USA), specifico per cellule macrofagiche umane.…”