Complement c3a and c5a induce different signal transduction cascades in endothelial cells

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Leukocytes utilize complement anaphylatoxins C3a and C5a for chemotaxis via G(i)-dependent pathways.
• Endothelial cells (HUVECs, dermal microvascular) express C3a and C5a receptors (C3aR, C5aR), but their functions are unclear.

Purpose of the Study:

• To investigate the functional roles of C3aR and C5aR signaling in human endothelial cells.
• To elucidate the distinct signaling pathways activated by C3a and C5a in endothelial cells compared to leukocytes.

Main Methods:

• Stimulation of endothelial cells with C3a and C5a.
• Analysis of cytoskeletal changes (actin stress fibers, cell retraction) and paracellular permeability.
• Pertussis toxin (PT) sensitivity assays, Rho activation assays, and kinase/receptor inhibition studies (PI3K, src, EGFR).
• Western blotting for EGFR phosphorylation and eosinophil transmigration assays.

Main Results:

• C3aR activation induced transient actin stress fibers via Rho, independent of PT, suggesting G(alpha12/13) coupling.
• C5aR activation caused delayed cytoskeletal changes, cell retraction, increased permeability, and eosinophil transmigration, sensitive to PT.
• C5a-induced responses were blocked by EGFR inhibitors and involved EGFR transactivation and phosphorylation, a novel pathway for endothelial C5aR signaling.

Conclusions:

• Endothelial C3aR and C5aR signal through distinct pathways, differing from leukocyte signaling.
• C5aR signaling in endothelial cells uniquely involves epidermal growth factor receptor (EGFR) transactivation, facilitating eosinophil transmigration.
• C3aR couples to G(alpha12/13), while C5aR utilizes a PT-sensitive cascade involving EGFR transactivation.