title: Effects of combined treatments with CTLA4-IG (abatacept), dexamethasone and methotrexate on cultured human macrophages.
abstract: OBJECTIVE
  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.
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy. mcutolo@unige.it.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cutolo
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Paolino
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Pizzorni
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Sulli
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Seriolo
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Marco Amedeo
authors.initials: MA
authors.lastname: Cimmino
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Montagna
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Soldano
authors.affiliation: Division of Clinical Immunology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Contini
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Italy.
authors.forename: Renata
authors.initials: R
authors.lastname: Brizzolara
mesh_terms: D000069594:Abatacept / Q000494:pharmacology*; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004359:Drug Therapy, Combination; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D016896:Treatment Outcome
chemical_list: D016207:Cytokines; D005938:Glucocorticoids; D007166:Immunosuppressive Agents; D000069594:Abatacept; D003907:Dexamethasone; D008727:Methotrexate