Differential ability of exogenous chemotactic agents to disrupt transendothelial migration of flowing neutrophils

Neutrophils migrate through endothelium using an ordered sequence of adhesi ve interactions and activating signals. To investigate the consequences of disruption of this sequence, we characterized adhesion and migration of neu trophils perfused over HUVEC that had been treated with TNF-alpha for 4 h a nd evaluated changes caused by exogenously added chemotactic agents. When H UVEC were treated with 2 U/ml TNF, flowing neutrophils adhered, with the ma jority rolling and relatively few migrating through the monolayer, If fMLP, IL-8, zymosan-activated plasma (a source of activated complement factor C5 a), epithelial cell-derived neutrophil-activating peptide (ENA-78), or grow th-regulating oncogene, GRO-alpha, was perfused over these neutrophils, the y stopped rolling and rapidly migrated over the monolayer, but did not pene trate it. When HUVEC were treated with 100 U/ml TNF, the majority of adhere nt neutrophils transmigrated. If neutrophils were treated with fMLP, IL-8, C5a, ENA-78, or GRO-alpha just before perfusion over this HUVEC, transmigra tion, but not adhesion, was abolished. However, when platelet-activating fa ctor was used to activate neutrophils, migration through HUVEC treated with 100 U/ml TNF was not impaired, and migration through HUVEC treated with 2 U/ml TNF was actually increased. Transmigration required ligation of CXC ch emokine receptor-2 on neutrophils, and differential desensitization of this receptor (e.g., by fMLP but not platelet-activating factor) may explain th e pattern of disruption of migration. Thus, transmigration may require pres entation of the correct activators in the correct sequence, and inappropria te activation (e.g., by systemic activators) could cause pathological accum ulation of neutrophils in the vessel lumen.