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FRI0175 CTLA4-IG (Abatacept), Dexamethasone and Methotrexate Combined Treatments: Effects on Cultured Human Macrophages
  1. M. Cutolo,
  2. S. Soldano,
  3. A.C. Trombetta,
  4. A. Sulli,
  5. B. Seriolo,
  6. M.A. Cimmino,
  7. S. Paolino,
  8. C. Pizzorni,
  9. P. Montagna,
  10. R. Brizzolara
  1. Research Laboratory and Academic Division of Clinical Rheumatology, DIMI, University of Genova, Genova, Italy

Abstract

Background The fusion protein CTLA4-Ig (abatacept), by interacting with the CD86 molecule on rheumatoid arthritis (RA) synovial macrophages, may induce in vitro 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.

Objectives 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.

Methods 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 (10-8 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.

Results After 3 hrs, ABAT alone significantly reduced IL-1β (p<0.01), TNFα (p<0.05) and IL-6 (p<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<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<0.01) only for IL-1β; while ABAT alone also induced a significant decrease but limited to TNFα (p<0.05). ICC showed, after 24 hrs, for all the tested treatments, a significant decrease for IL1β (p<0.05), IL6 (p<0.05) and TNFα (p<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<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<0.05).

Regarding MTX a significant inhibition of gene expression (p<0.01) was observed only after 3 hrs versus control.

Conclusions 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.

References

  1. Cutolo M et al. Arthritis Res Ther 2009, 11:176-85.

  2. Brizzolara R et al. Reumatismo 2011, 63:80-5.

  3. Brizzolara R et al. J Rheumatol 2013;40(5):738-40.

  4. Cutolo M et al. Clin Exp Rheumatol 2013;31(6):943-6.

Disclosure of Interest 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

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