Abstract
Purpose :
Dry eye disease (DED) is a common, debilitating inflammatory eye disease affecting normal vision and quality of life. Our previous studies have demonstrated an increase in ocular surface-associated neutrophil adhesion and formation of neutrophil extracellular traps (NETs) on the ocular suface of patients with DED. The goal of this study was to test the efficacy of pooled human immune globulins against neutrophil adhesion to corneal epithelial cells and formation of NETs.
Methods :
Human neutrophils were isolated from whole blood of healthy volunteers by immunobead magnetic cell separation (Miltenyi Biotec) and maintained in RPMI1640 media supplemented with 10 mM d-glucose, 1 mM sodium pyruvate and 2 mM l-Glutamine. Neutrophil activation in vitro was determined by adhesion of neutrophils to human corneal epithelial cells (HCE-T, Riken BioResource Center, Japan) quantification of NET formation following stimulation with either histone-4 anti-citrullinated protein antibody (H4R3 ACPA, 100 ng/mL, Abcam) or phorbol 12-myristate 13-acetate (PMA, 20 nM, Millipore Sigma, St. Louis, MO) in the presence or absence of pooled human immune globulins (Flebogamma® DIF 5%; Grifols, S.A.). For adhesion assays, neutrophils were labeled with 1 µM calcein AM (Thermo Fischer), while NETs were identified by SYTOXTM Green to label extracellular DNA.
Results :
H4R3 ACPA resulted in a ~2-fold increase of neutrophil adhesion and NET formation, while PMA resulted in an over 100-fold increase in neutrophil activation. Pooled human immune globulins (0.1 – 5%) resulted in a dose-dependent decrease against H4R3 ACPA- and PMA-induced neutrophil activation. Interestingly, only high doses of pooled human immune globulins (5% Flebogamma®) resulted in a statistically significant decrease in neutrophil activation.
Conclusions :
Pooled human immune globulins exert potent dose-dependent protection against H4R3 ACPA- and PMA-induced neutrophil activation in vitro. Notably, only high concentrations of pooled human immune globulins (5%) resulted in a significant decrease of neutrophil adhesion to corneal epithelial cells, consistent with prior studies in different organ system. Together with favorable ocular safety and tolerability studies, these data support the further development of Flebogamma® 5% DIF for the treatment of the signs and symptoms of DED.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.