Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
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.
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.
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).
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.
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 10). https://acrabstracts.org/abstract/neutralizing-effect-of-anti-infliximab-antibodies-on-infliximab-stimulated-human-coronary-artery-endothelial-cells/. Accessed April 13, 2021.
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