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Abstract Number: 996

Neutralizing Effect of Anti-Infliximab Antibodies on Infliximab-Stimulated Human Coronary Artery Endothelial Cells

Manca Ogrič1, Katjuša Mrak Poljšak1, Katja Lakota1,2, Polona Žigon1, Sonja Praprotnik1, Saša Čučnik1,3 and Snezna Sodin Semrl1,2, 1University Medical Centre Ljubljana, Department of Rheumatology, Ljubljana, Slovenia, 2University of Primorska, Faculty of Mathematics, Natural Science and Information Technology, Koper, Slovenia, 3University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia

Meeting: 2018 ACR/ARHP Annual Meeting

Keywords: cytokines, endothelial cells and infliximab

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Session Information

Date: Monday, October 22, 2018

Session Title: Cytokines and Cell Trafficking Poster

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose:

As a consequence of endothelial dysfunction, patients with rheumatoid arthritis (RA) have an increased risk of atherosclerosis and early development of cardiovascular disease. Serum amyloid A (SAA), tumour necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) up-regulated in the sera of RA patients were previously reported to activate human coronary artery endothelial cells (HCAEC), however their combined effects are unclear. Furthermore, the effect of anti-TNF-α drugs, such as infliximab, in the presence and absence of their anti-infliximab antibodies, has not yet been elucidated.

In our study, we aimed to investigate the combined effects of TNF-α, IL-1β, SAA, infliximab and anti-infliximab antibodies on IL-6 released levels in HCAEC.

Methods:

Primary HCAEC (Lonza), passage 5, were grown to confluency in 5% FBS/EGM-2M medium, serum starved for 2 hours and incubated with human recombinant cytokines (SAA1/2 (Peprotech, 500nM), TNF-α (Thermo Fisher Scientific, 2.5 ng/mL) and IL-1β (Thermo Fisher Scientific, 1 ng/mL)), infliximab, anti-infliximab antibodies and their combinations. After 24 hours, supernatants were collected, centrifuged, aliquoted and frozen at -20°C.

Infliximab was used at a final concentration of 10 μg/mL, in combination with the cytokines. For the neutralizing effect of anti-infliximab antibodies (final concentration 0.55 μg/mL) on infliximab (final concentration 0.1 μg/mL), polyclonal anti-infliximab antibodies were purified by affinity chromatography from sera samples of 2 patients with chronic rheumatic diseases, who exhibited positive levels of anti-infliximab antibodies, as previously determined by an in-house competitive and bridging ELISA. IL-6 was measured by ELISA (Invitrogen). One-way ANOVA was used for statistical analysis.

Results:

Triple stimulation of HCAEC with TNF-α/IL-1β/SAA significantly and synergistically elevated the release of IL-6 levels in cell supernatants (3-fold above IL-1β alone). Double stimulation with IL-1β/SAA, TNF-α/SAA, as well as TNF-α/IL-1β and IL-1β alone, also led to significantly higher levels of IL-6, while TNF-α alone did not increase IL-6 levels.

Infliximab was effective in lowering released IL-6 levels in the TNF-α/IL-1β double treatment, however the strongest inhibition was observed in TNF-α/IL-1β/SAA triple-stimulated HCAEC, where it significantly reduced IL-6 released levels by around 50%.

Anti-infliximab antibodies significantly restored IL-6 released levels from HCAEC treated with infliximab and TNF-α/IL-1β/SAA (complete neutralization of the infliximab inhibition).

Conclusion:

TNF-α, IL-1β and SAA synergistically elevated IL-6 release in supernatants of HCAEC, with infliximab substantially inhibiting its levels. Isolated polyclonal anti-infliximab antibodies were capable of neutralizing infliximab, in the presence of TNF-α/IL-1β/SAA, thereby promoting chronic inflammation in HCAEC.


Disclosure: M. Ogrič, None; K. Mrak Poljšak, None; K. Lakota, None; P. Žigon, None; S. Praprotnik, None; S. Čučnik, None; S. Sodin Semrl, None.

To cite this abstract in AMA style:

Ogrič M, Mrak Poljšak K, Lakota K, Žigon P, Praprotnik S, Čučnik S, Sodin Semrl S. Neutralizing Effect of Anti-Infliximab Antibodies on Infliximab-Stimulated Human Coronary Artery Endothelial Cells [abstract]. Arthritis Rheumatol. 2018; 70 (suppl 9). https://acrabstracts.org/abstract/neutralizing-effect-of-anti-infliximab-antibodies-on-infliximab-stimulated-human-coronary-artery-endothelial-cells/. Accessed January 27, 2023.
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