A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses

We previously reported an unusual carboxylated modification on N-glycans is olated from whole bovine lung. We have now raised Ig: mAbs against the modi fication by immunization with biotinylated aminopyridine-derivatized glycan s enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides, One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopep tides and other multicarboxylated molecules, but not by glycopeptides in wh ich the carboxylate groups were modified. The Ab recognized an epitope cons titutively expressed on bovine, human, and other mammalian endothelial cell s. Stimulated, but not resting, neutrophils bound to immobilized bovine lun g glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murin e model of peritoneal inflammation. This inhibition of cell trafficking cor related with the increased sequestration but reduced transmigration of leuk ocytes that were found to be adherent to the endothelium of the mesenteric microvasculature, Taken together, these results indicate that these novel c arboxylated N-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activat ed neutrophils.