Background/Purpose

RA is an autoimmune rheumatic disease characterised by ACPA, endothelial dysfunction, platelet activation, synovial hyperplasia and hypoxia in affected joints. ACPA recognise citrullinated proteins, generated by the activity of peptidyl arginine deiminase (PAD)-2/4, however the source of these autoantigens is unknown. Neutrophil extracellular traps (NETs) play a critical role in the innate immune response and have been identified in serum and synovial fluid of patients with RA. NETs require PAD-4 for their generation and are also a source of citrullinated proteins. NET formation is enhanced by integrin engagement, interactions with activated platelets, and other stimuli but the effects of hypoxia upon their generation are unknown. Therefore, we hypothesise that hypoxia modulates the cellular interactions leading to enhanced NETosis and the externalisation of citrullinated autoantigens in RA.

Methods

Human umbilical cord endothelial cells (HUVEC) were grown in hypoxia (1% oxygen, equivalent to 7.6 mmHg) to mimic conditions found in the RA joint or normoxia (21% oxygen, 159.6 mmHg) in the presence or absence of TNF-α or lipopolysaccharide (LPS).  Expression of PAD-4 and citrullinated histone H3, a PAD-4 target, were measured by western blot, and levels of surface adhesion molecules, intercellular adhesion molecule (ICAM)-1, ICAM-2, vascular cell adhesion molecule (VCAM)-1 and E selectin, were measured by FACs. Neutrophils were isolated from whole blood donated by healthy volunteers. Cell adhesion assays were performed in which binding of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) labelled neutrophils to HUVEC monolayers was measured using a fluorescent plate reader. A capture ELISA was established and validated to compare NET release between experimental conditions.

Results

Culture of HUVEC under hypoxia increased PAD-4 expression by 13.5% (1.2 fold) with levels of citrullinated histone H3 elevated by 1415.3% (15 fold). Hypoxia modulated both basal expression of ICAM-1, ICAM-2, VCAM-1 and E selectin as well as on stimulation with TNF-α or LPS. Hypoxia also increased neutrophil adhesion to HUVEC monolayers from 3+/-3.1% to 47+/-12.0% of total cells added (p=0.0002). NET formation of phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils was elevated under hypoxia compared to unstimulated controls (49.6% vs. 58.4%, p<0.01). Co-culture experiments demonstrated enhanced NETosis of PMA-stimulated neutrophils cultured with HUVEC compared to neutrophils cultured alone under normoxia (p<0.05).

Conclusion

We have shown that hypoxia modulates: PAD-4 expression; citrullination of histone H3; adhesion molecule expression in HUVEC; neutrophil adherence to HUVEC monolayers; and NET release. Furthermore, co-culture of neutrophils with HUVEC under normoxia elevates NET production on stimulation. Given that these processes are all relevant to the pathogenesis of citrullinated antigens in RA, further experiments are currently underway to dissect the modulatory effects of hypoxia on NETosis and investigate the effects of ACPA positive RA-IgG upon these cellular conditions.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/hypoxia-modulates-peptidyl-arginine-deiminase-4-activity-and-neutrophil-extracellular-trap-formation/