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Effects of combined treatments with CTLA4-IG (abatacept), dexamethasone and methotrexate on cultured human macrophages
M. Cutolo1, S. Paolino2, C. Pizzorni3, A. Sulli4, B. Seriolo5, M.A. Cimmino6, P. Montagna7, S. Soldano8, P. Contini9, R. Brizzolara10
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy. mcutolo@unige.it
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
- Division of Clinical Immunology, Department of Internal Medicine, University of Genova, Italy.
- Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
CER8875
2016 Vol.34, N°3
PI 0500, PF 0506
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PMID: 26940199 [PubMed]
Received: 19/08/2015
Accepted : 14/12/2015
In Press: 03/03/2016
Published: 30/05/2016
Abstract
OBJECTIVES:
To evaluate the anti-inflammatory effect of CTLA4-Ig (abatacept) and dexamethasone (DEX) monotreatment versus their combination and adding methotrexate (MTX) on cultured human macrophages.
METHODS:
THP-1 cells, activated into macrophages (PMA 0.05 μg/ml), were cultured for 3 and 24 hrs with CTLA4-Ig (500 μg/ml), DEX (10-8 M), MTX (0.05 μg/ml), and CTLA4-Ig combined with DEX or CTLA4-Ig combined with DEX plus MTX. CTLA4-Ig/CD86 interaction was evaluated by FACS analysis. Quantitative real time-PCR (qRT-PCR), immunocytochemistry (ICC) and immunoassay (ELISA) analysis for inflammatory cytokine (IL-1β, TNF-α, IL-6) expression were performed.
RESULTS:
FACS analysis showed in macrophages treated with CTLA4-Ig alone, CTLA4-Ig-DEX and CTLA4-Ig-DEX-MTX a CD86 decrease of almost 35%, versus untreated cells (CNT). After 3 hrs, macrophages treated with DEX alone or with CTLA4-Ig-DEX or CTLA4-Ig-DEX-MTX showed a significant reduction (p<0.05) for all cytokines gene expression, that was still significant for IL-1β after 24 hrs (p<0.05). After 3 hrs, CTLA4-Ig alone significantly (p<0.05) reduced all cytokine genes; however, after 24 hrs still evident only for TNF-α (p<0.05). After 24 hrs CTLA4-Ig-DEX induced a significant decrease of gene expression (p<0.05) for TNF-α and IL-6, whereas CTLA4-Ig-DEX-MTX induced a decrease (p<0.05) limited to IL-6, versus CNT. Finally, ICC showed, after 24 hrs of CTLA4-Ig-DEX or CTLA4-Ig-DEX-MTX treatment a reduction (p<0.05) of IL-1β and IL-6 expression, versus CNT; DEX alone reduced only IL-1β (p<0.05). ELISA analysis confirmed these results.
CONCLUSIONS:
CTLA4-Ig-DEX and CTLA4-Ig-DEX-MTX combined treatments, decreased at any level the inflammatory cytokine expression more efficiently then monotreatments on activated cultured human macrophages.