Abstract / Summary
Background: Methotrexate (MTX) is commonly used in patients with autoimmune disorders with the therapeutic objective of inducing immunosuppression predominantly through the modulation of T-lymphocyte activity. Additional studies suggest that the regulation of polymorphonuclear neutrophils (PMNs) may also play a significant role in disease modification. However, the molecular mechanism underlying this effect remains incompletely understood. Moreover, most studies investigating the interaction between MTX and PMNs have relied on centrifugation for cell isolation, a method meanwhile known to alter PMN behavior and activation status itself.
Objectives: The aim of the study was to investigate the effects of MTX on PMN function-including reactive oxygen species (ROS) production, NETosis (Neutrophil Extracellular Traps), and migration-using live-cell imaging, as well as surface epitopes and the respiratory burst assessed by flow cytometry. This comprehensive set of experiments examining the interaction between MTX and PMNs provides a previously unreported level of insight.
Methods: PMNs from healthy donors were isolated using a sedimentation procedure with 10% Gelafundin and subsequently incubated with 0, 0.005, 0.5, or 5 mmol/L MTX for downstream experiments. Live-cell imaging was used to assess neutrophil migration, NETosis, and ROS production, while flow cytometry was conducted to evaluate the activation markers CD11b, CD66b, and CD62L, as well as the surface marker LOX-1 (lectin-type oxidized LDL receptor-1) and ROS production.
Results: Live-cell imaging demonstrated a significant inhibition of chemotactic PMN migration at all MTX concentrations compared to controls. In contrast, neither ROS production nor NETosis could be detected under the same conditions. Flow cytometric analysis revealed a significant upregulation of LOX-1 surface expression, while the activation markers CD11b, CD62L, and CD66b remained unaltered. Furthermore, an increase in ROS production during the respiratory burst could not be detected.
Conclusion: These results indicate that MTX reduces the chemotaxis of most naive PMNs. In addition, MTX appears to induce an upregulation of LOX-1. Future studies should further investigate the impact of LOX-1 on PMN function.