FRI0175 CTLA4-IG (Abatacept), Dexamethasone and Methotrexate Combined Treatments: Effects on Cultured Human Macrophages
Authors: M. Cutolo, S. Soldano, A.C. Trombetta, A. Sulli, B. Seriolo, M.A. Cimmino, S. Paolino, C. Pizzorni, P. Montagna, R. Brizzolara
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background</h3> The fusion protein CTLA4-Ig (abatacept), by interacting with the CD86 molecule on rheumatoid arthritis (RA) synovial macrophages, may induce <i>in vitro</i> reverse intracellular signaling with anti-inflammatory effects, through the involvement of the NFkB intracellular pathway [1-4]. In RA the combination of abatacept (ABAT) and glucocorticoids (GC) or/and methotrexate (MTX) allows to obtain better clinical improvement compared to ABAT monotherapy. <h3>Objectives</h3> To evaluate the anti-inflammatory effect of ABAT and dexamethasone (DEX) monotreatment versus their combination and plus MTX at gene and protein expression level in cultured human activated macrophages. <h3>Methods</h3> THP-1 cells, activated into macrophages (PMA 0.05 μg/ml; 24 hrs), were cultured for 3, 24 and 48 hrs with ABAT (500 μg/ml) or DEX (10<sup>-8</sup> M) alone, DEX combined with ABAT, and DEX with ABAT plus MTX (0.05 μg/ml). Then IL-1β, TNFα and IL-6 gene and protein expression was evaluated by quantitative real time-PCR (qRT-PCR) (after 3 and 24 hrs) and by immunocytochemistry (ICC) analysis (after 24 and 48 hrs). Untreated cells were used as controls. <h3>Results</h3> After 3 hrs, ABAT alone significantly reduced IL-1β (p&lt;0.01), TNFα (p&lt;0.05) and IL-6 (p&lt;0.01) gene expression (qRT-PCR), in addition, macrophages treated with DEX alone or DEX-ABAT or DEX-ABAT-MTX combined treatments, showed a significant reduction (p&lt;0.01) for the gene expression of all assayed cytokines, compared to the control. After 24 hrs, DEX alone or DEX-ABAT or DEX-ABAT-MTX combined treatments still showed the most significant reduction in gene expression (p&lt;0.01) only for IL-1β; while ABAT alone also induced a significant decrease but limited to TNFα (p&lt;0.05). ICC showed, after 24 hrs, for all the tested treatments, a significant decrease for IL1β (p&lt;0.05), IL6 (p&lt;0.05) and TNFα (p&lt;0.05) compared to the controls. After 48 hrs, ICC still showed a decrease for IL-1β and TNFα protein expression not significant after DEX alone and significant after DEX-ABAT (p&lt;0.05), compared to untreated cells (controls), while IL6 protein expression resulted unchanged after ABAT treatment, compared to the control. Interestingly, after 48 hrs, ABAT alone significantly reduced IL-1β and TNFα production (p&lt;0.05). Regarding MTX a significant inhibition of gene expression (p&lt;0.01) was observed only after 3 hrs versus control. <h3>Conclusions</h3> DEX-ABAT or DEX-ABAT-MTX combined treatments, induced a rapid anti-inflammatory effect on cultured human macrophages, by decreasing proinflammatory cytokine production. The reduction, was already significant after 3 hrs at gene level and after 24/48 hrs at protein level. Both combinations seem more efficient then monotreatment on cytokine gene expression, with stronger effects for DEX-ABAT at least at 3 hours. These effects could be implied in the RA treatment. <h3>References</h3> Cutolo M et al. Arthritis Res Ther 2009, 11:176-85. Brizzolara R et al. Reumatismo 2011, 63:80-5. Brizzolara R et al. J Rheumatol 2013;40(5):738-40. Cutolo M et al. Clin Exp Rheumatol 2013;31(6):943-6. <h3>Disclosure of Interest</h3> M. Cutolo Grant/research support from: The study was partially supported by research grant (university funds) by Bristol Myers Squibb and the compound for the experiments was provided by Bristol Myers Squibb., S. Soldano: None declared, A. C. Trombetta: None declared, A. Sulli: None declared, B. Seriolo: None declared, M. A. Cimmino: None declared, S. Paolino: None declared, C. Pizzorni: None declared, P. Montagna: None declared, R. Brizzolara: None declared