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AB0082 CTLA4-IG/CD86 Interaction on Cultured Human Endothelial Cells: Evaluation of VEGF-R and ICAM-1 Protein Expression
  1. M. Cutolo1,
  2. P. Montagna1,
  3. S. Soldano1,
  4. B. Seriolo1,
  5. P. Contini2,
  6. B. Villaggio3,
  7. R. Brizzolara1
  1. 1Research Laboratory and Academic Division of Clinical Rheumatology, DIMI, University of Genova
  2. 2Division of Clinical Immunology, Department of Internal Medicine, University of Genova
  3. 3Clinical Academic Unit of Nephrology, Department of Internal Medicine, University of Genova, Genova, Italy

Abstract

Background Endothelial cell (EC) dysfunction and angiogenesis are involved in synovitis in established rheumatoid arthtritis (RA) [1]. Some studies reported that the ECs express the repertoire of costimulatory molecules including CD86 (B7.2), for adequate T cell interaction/activation [2]. Our recent data showed that the fusion protein CTLA4-Ig (abatacept), used as biological agent in RA therapy, already interacts with the CD86 molecule expressed in synovial cells [3,4].

Objectives In the present study CTLA4-Ig/CD86 interaction and VEGF-R and ICAM-1 protein expression was evaluated in vitro on activated ECs.

Methods ECs (human microvascular endothelial cells, HMVEC, Lonza, Switzerland), after 7 days of culture, were induced by γ-IFN treatment (for 48 hours, 500 U/ml) to express CD86 molecules. Then, the cells were treated for 24 hours with CTLA4-Ig (10, 100, 500 μg/ml) and the protein expression of CD86, VEGF-R and ICAM-1 were evaluated by flow cytometric analysis (FACS). In addition, western blot analysis (WB) for VEGF-R and ICAM-1 protein expression were performed.

Results FACS analysis showed that, after CTLA4-Ig treatment (10, 100, 500 μg/ml), activated ECs decreased their CD86-positivity (66%, 59% and 51%, respectively), compared to untreated cells (68%), suggesting a CTLA4-Ig/CD86 interaction and masking on their surface. Therefore, almost all the activated ECs expressed VEGF-R (79%) and all activated ECs strongly expressed ICAM-1 (99%). After 24 hours of CTLA4-Ig treatment, the cells showed a decrease dose-dependent in the mean fluorescence value for ICAM-1, while VEGF-R positivity resulted unchanged. WB analysis for VEGF-R and ICAM-1 protein expression also showed a marked decrease in activated ECs treated with CTLA4-Ig at 500 μg/ml.

Conclusions The results observed at FACS analysis suggest an interaction between CTLA4-Ig and CD86 on activated ECs (expressing the CD86 molecule). In addition, a modulation in the expression of molecules relevant for the inflammatory and angiogenetic processes was observed. ICAM-1 protein expression shows on activated ECs a dose-dependent decrease after CTLA4-Ig treatment, at least for the higher dose treatment. Otherwise, VEGF-R protein expression showed a decrease only observed in WB analysis. In conclusion, ECs might be considered a further target for CTLA4-Ig in inflammatory conditions such as in presence of synovitis.

References

  1. Marrelli A et al. Autoimmun Rev. 2011;10(10):595-8.

  2. Kreisel D et al. J Immunol 2002;169(11):6154-61.

  3. Brizzolara R et al. Reumatismo. 2011;63(2):80-54.

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

Disclosure of Interest M. Cutolo Grant/research support: Bristol Myers Squibb, P. Montagna: None declared, S. Soldano: None declared, B. Seriolo Grant/research support: Bristol Myers Squibb, P. Contini: None declared, B. Villaggio: None declared, R. Brizzolara: None declared

DOI 10.1136/annrheumdis-2014-eular.3563

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