Endothelial oxidative stress activates the lectin complement pathway: role of cytokeratin 1

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Oxidative stress is known to increase endothelial mannose-binding lectin (MBL) binding and activate the lectin complement pathway (LCP).
• The precise molecular mechanism linking oxidative stress to MBL binding on endothelial cells remained unclear.
• Intermediate filaments have previously been implicated in antibody-independent classical complement pathway activation.

Purpose of the Study:

• To investigate if oxidative stress upregulates cytokeratin 1 (CK1) expression in human umbilical vein endothelial cells (HUVECs).
• To determine if MBL binding to CK1 mediates the activation of the LCP following oxidative stress.
• To elucidate the role of endothelial CK1 in oxidative stress-induced complement activation.

Main Methods:

• Hypoxic HUVECs were reoxygenated to induce oxidative stress.
• CK1 mRNA and membrane protein expression were quantified using in situ hybridization and ELISA/confocal microscopy.
• MBL and C3 deposition on purified CK1 and HUVECs were assessed using ELISA and confocal microscopy, with and without MBL inhibition or anti-CK1 antibodies.

Main Results:

• Reoxygenation significantly increased CK1 mRNA and membrane protein expression in HUVECs.
• MBL and C3 deposition on purified CK1 was attenuated by N-acetyl-D-glucosamine and anti-MBL antibodies.
• CK1 and MBL were co-immunoprecipitated, and anti-cytokeratin Fab fragments reduced endothelial MBL and C3 deposition post-oxidative stress.

Conclusions:

• Endothelial oxidative stress enhances CK1 expression, leading to increased MBL binding and C3 deposition.
• Inhibition of MBL effectively reduces CK1-induced complement activation.
• Anti-cytokeratin antibodies significantly attenuate complement deposition on endothelial cells after oxidative stress, suggesting MBL-CK1 interaction mediates LCP activation.