Cultured human endothelial cells elaborate neutrophil chemoattractants

Abstract

The recruitment of circulating neutrophils to acutely injured lungs might be regulated by vascular endothelial cells. Conditioned media from confluent monolayers of endothelial cells derived from human umbilical veins were assayed for neutrophil chemotactic activity by the Boyden chamber technique. Among 20 confluent monolayers tested, 14 (70%) elicited chemotactic activity. The generation of chemotactic activity was time dependent. In four experiments, the chemotactic index at 6 hours of incubation was 3.7 +/- 0.4, at 24 hours was 5.0 +/- 0.5, and at approximately 48 hours was 6.6 +/- 0.6. Chemotactic activity was detectable in dilutions of 1:4 to 1:8. To characterize the factors in conditioned media responsible for enhanced neutrophil migration, pooled conditioned media from confluent endothelial cell monolayers eliciting maximal chemotactic activity (chemotactic index = 7.46 on pooled sample) were chromatographed on a Biogel A-0.5m column. Two column fractions corresponding to molecular weights of 35,000 and approximately 1500 daltons enhanced neutrophil migration. No migration-enhancing column effluents were observed from unconditioned medium. Both of the active column fractions from conditioned media were found to be chemotactic by checkerboard analysis. Activity in the 35,000-dalton peak was abolished by either treatment with Pronase or heat (56 degrees C X 30 minutes) but was not extracted into chloroform-methanol. Conversely, chemotactic activity in the 1500-dalton peak resisted heat and proteolysis but was extractable into chloroform-methanol. These data indicate that cultured human endothelial cells generate neutrophil chemoattractants, and that at least two chemoattractants, a 35,000-dalton protein and a 1500-dalton lipid, are generated. Our findings suggest that endothelial cells have the capacity to recruit neutrophils to vascular surfaces and may thereby initiate or modulate the inflammatory response.