Background/Purpose: Tumor-necrosis factor (TNF)-α plays a key role in the pathophysiology of rheumatoid arthritis (RA) and membrane bound TNFα is sufficient to induce arthritis in mice [1]. Zhang [2] demonstrated membrane bound TNFα on extracellular vesicles (EVs) derived from RA synovial fibroblasts. EVs are stable in body fluids and mediate intercellular communication over short and long distances. In this study we want to determine whether TNFα is also present on circulating EVs from RA patients and not in healthy individuals.

Methods: pEVs were obtained from 28 RA patients and 24 healthy controls (HC) by size exclusion chromatography. Protein content was measured by micro-BCA, size and concentration by Nanoparticle Tracking. TNFα was detected by bead-based multiplex immunoassays (BBI) and flowcytometry (FC). To control for specificity, pEVs were preincubated with anti-TNFα (etanercept and certolizumab pegol). Immunoglobulin M rheumatoid factor (IgM-RF) was measured in pEVs by ELISA. RF+ and RF- pEVs were preincubated with Protein L beads to bind RF-IgM before TNFα was measured by FC.

Results: Particle size and protein content per particle were significantly higher in RA-pEVs as compared to HC (154nm, 484fg and 116nm, 216fg, resp) while particle concentration was not statistically different between RA patients and HC (0.89, 2.59 x10¹⁰/ml, resp). In 13 out of 28 RA patients TNFα (220 pg/ml) was detectable in pEVs by BBI whereas in HC TNFα was undetectable. Presence of TNFa on RA-pEVs was confirmed by FC. Preincubation of pEVs with anti-TNFα antibodies (etanercept) fully blocked TNFα detection. Unexpected, after preincubation with anti-TNFα Fab fragments (certolizumab pegol) TNFα was still detectable which suggested aspecific binding of the TNFa detection antibody. By ELISA IgM-RF was detectable on pEVs in 9 out of 13 RA patients. By preincubation with Protein-L coupled magnetic beads, to scavenge RF, TNFα levels on pEVs isolated from RF⁺ RA patients were reduced to background.

Conclusion: This study shows for the first time that IgM-RF is present on pEVs in a subpopulation of RA patients and this impedes with immunodetection of TNFα. To obtain conclusive evidence for the presence of TNFα other laboratory techniques are needed. However, the presence of RF on EVs could be important in the immunopathophysiology of RA by directing putative immunoregulatory and potentially inflammatory EVs to sites of immune-complex formation.

1: Keffer. EMBO J. (1991)

2: Zhang. J Immunol (2006)

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-presence-of-igm-rheumatoid-factor-impede-immunodetection-of-tnfa-on-circulating-extracellular-vesicles-obtained-from-rheumatoid-arthritis-patients/