Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose
CTLA4-Ig (abatacept) is employed as biological agent in rheumatoid arthtritis (RA) treatment and interacts with the costimulatory molecule CD86 expressed by different cells involved in synovitis [1,2]. The presence of CD86 has been shown on endothelial cell (EC) surface and their dysfunction is involved in the angiogenetic processes characterizing RA synovitis [3,4]. Since, ICAM1 (intercellular adhesion molecule 1) and VEGFR-2 (vascular endothelial growth factor receptor 2) are relevant molecules for the inflammatory and angiogenetic processes, their expression was evaluated in vitro on activated ECs at protein and gene expression level, following CTLA4-Ig treatment.
Methods
ECs (human microvascular endothelial cells, HMVECs, Lonza, Switzerland) were induced by gamma-IFN (500 U/ml for 48 hours) to activate and to express CD86. The cells were then treated for 24 hrs with CTLA4-Ig (10, 100, 500 micrograms/ml), and the protein expression levels for CD86, ICAM1 and VEGFR-2 were evaluated by flow cytometric analysis (FACS). After 3 and 24 hrs of CTLA4-Ig treatment, the protein expression levels of both ICAM1 and VEGFR-2 were also evaluated by Western blot analysis (WB) and their respective gene expression was evaluated by quantitative real time PCR (qRT-PCR). The statistical analysis was performed using the Mann-Whitney non-parametric t test.
Results
Activated ECs, after CTLA4-Ig treatment (10, 100, 500 μg/ml; 24 hrs), decreased their CD86-positivity at FACS by 66%, 59% and 51%, respectively, versus 68% of untreated cells (cnt), suggesting CTLA4-Ig/CD86 interaction and CD86 masking. All activated ECs, analysed by FACS, strongly expressed ICAM1 (99%) and VEGFR-2 (79%). Interestingly, after 24 hrs of CTLA4-Ig treatment, ECs showed a dose-dependent decrease in the mean fluorescence intensity for ICAM1, while VEGFR-2 positivity resulted unchanged. However, WB showed a significant decrease after CTLA4-Ig 500 μg/ml treatment for both ICAM1 and VEGFR-2 (p<0.05 at 3 and 24 hrs) while a significant decrease after CTLA4-Ig 100 μg/ml treatment was observed only for VEGFR-2 at 3 hrs (p<0.05). Similarly, qRT-PCR showed a significant decrease after CTLA4-Ig 500 μg/ml treatment for VEGFR-2 (p<0.05 at 3 and 24 hrs) and a significant decrease after CTLA4-Ig 100 μg/ml treatment for VEGFR-2 at 3 hrs (p<0.05). qRT-PCR at 3 hrs seemed to late to detect ICAM1 changes.
Conclusion
Results suggest a significant modulation by CTLA4-Ig of the gene expression and protein synthesis for ICAM1 and VEGFR-2 in cultured human ECs, which seems mainly due to the interaction between CTLA4-Ig and CD86 on activated ECs, possibly involving NFkB pathway downregulation, as previously reported [2]. Therefore, in the presence of chronic inflammatory reaction, such as in RA synovitis, ECs might be considered among other cells a further possible target for CTLA4-Ig modulation.
References. 1. Cutolo M et al. Autoimmun Rev. 2013;12:758-67 2. Cutolo M et al. Clin Exp Rheumatol. 2013;31:943-6. 3. Kreisel D et al. J Immunol 2002;169:6154-61. 4. Marrelli A et al. Autoimmun Rev. 2011;10:595-8.
Disclosure:
M. Cutolo,
Bristol Myers Squibb,
2;
P. Montagna,
None;
S. Soldano,
None;
P. Contini,
None;
B. Villaggio,
None;
A. Sulli,
None;
S. Paolino,
None;
C. Pizzorni,
None;
B. Seriolo,
None;
G. Zampogna,
None;
M. A. Cimmino,
None;
R. Brizzolara,
None.
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