Double-Hit-Induced Leukocyte Extravasation Driven by Endothelial Adherens Junction Destabilization

Area of Science:

• Immunology
• Cell Biology
• Vascular Biology

Background:

• Endothelial cells respond to inflammatory stimuli.
• Leukocyte extravasation and vascular leakage are key inflammatory processes.
• These processes are typically considered uncoupled due to distinct spatiotemporal patterns.

Purpose of the Study:

• To investigate the combined effect of lipopolysaccharide (LPS) and histamine on endothelial cells.
• To determine the mechanisms underlying synergistic neutrophil transendothelial migration (TEM).
• To evaluate junctional linearity as a readout for anti-inflammatory compounds.

Main Methods:

• Primary human endothelial cells were treated with LPS followed by histamine (double-hit).
• Neutrophil TEM was assessed using physiological flow assays.
• Endothelial barrier function, adhesion molecule expression, and Weibel-Palade body release were analyzed.
• Junctional remodeling and linearity were examined.

Main Results:

• The double-hit approach synergistically enhanced neutrophil TEM compared to individual stimuli.
• This enhancement was independent of endothelial barrier changes, adhesion molecules, or Weibel-Palade bodies.
• Neutrophil TEM increase correlated with junctional remodeling.
• Compounds stabilizing endothelial junctions counteracted the double-hit effect.

Conclusions:

• Histamine can have significant secondary effects on neutrophil TEM when combined with inflammatory stimuli like LPS.
• The synergistic effect is mediated by endothelial cytoskeleton modifications and junctional remodeling.
• Junctional linearity is a more predictive marker than endothelial resistance for assessing anti-inflammatory compounds.