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Endothelial oxidative stress activates the lectin complement pathway: role of cytokeratin 1
C D Collard1, M C Montalto, W R Reenstra
1Department of Anesthesiology, Perioperative, and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Oxidative stress increases endothelial mannose-binding lectin (MBL) binding and activates the lectin complement pathway (LCP). However, the molecular mechanism of MBL binding to the endothelium after oxidative stress is unknown. Intermediate filaments have been previously reported to activate the classical complement pathway in an antibody-independent manner. We investigated whether oxidative stress increases human umbilical vein endothelial cell (HUVEC) cytokeratin 1 (CK1) expression and activates the LCP via MBL binding to CK1. Reoxygenation (3 hours, 21% O(2)) of hypoxic HUVECs (24 hours, 1% O(2)) significantly increased CK1 mRNA (in situ hybridization) and membrane protein expression [enzyme-linked immunosorbent assay (ELISA)/confocal microscopy]. Incubating human serum (HS) with N-acetyl-D-glucosamine or anti-human MBL monoclonal antibody attenuated MBL and C3 deposition on purified CK1 (ELISA). CK1 and MBL were co-immunoprecipitated from hypoxic HUVECs reoxygenated in HS. Treatment with anti-human cytokeratin Fab fragments attenuated endothelial MBL and C3 deposition after oxidative stress (ELISA/confocal microscopy). We conclude that: 1) endothelial oxidative stress increases CK1 expression, MBL binding, and C3 deposition; 2) inhibition of MBL attenuates purified CK1-induced complement activation; and 3) anti-human cytokeratin Fab fragments attenuate endothelial MBL and C3 deposition after oxidative stress. These results suggest that MBL binding to endothelial cytokeratins may mediate LCP activation after oxidative stress.
Insights
Oxidative stress elevates cytokeratin 1 (CK1) on endothelial cells, promoting mannose-binding lectin (MBL) attachment and activating the lectin complement pathway (LCP). This interaction is key to understanding endothelial complement activation.
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.