Neutrophil-derived glutamate regulates vascular endothelial barrier function.
Endothelial barrier function is altered by the release of soluble polymorphonuclear leukocyte (PMN)-derived mediators during inflammatory states. However, endogenous pathways to describe such changes are only recently appreciated. Using an in vitro endothelial paracellular permeability model, cell-free supernatants from formylmethionylleucylphenylalanine-stimulated PMNs were observed to significantly alter endothelial permeability. Biophysical and biochemical analysis of PMN supernatants identified PMN-derived glutamate in modulating endothelial permeability. Furthermore, novel expression of metabotropic glutamate receptor 1 (mGluR1), mGluR4, and mGluR5 by human brain and dermal microvascular endothelial cells was demonstrated by reverse transcription-PCR, in situ hybridization, immunofluorescence, and Western blot analysis. Treatment of human brain endothelia with glutamate or selective, mGluR group I or III agonists resulted in a time-dependent loss of phosphorylated vasodilator-stimulated phosphoprotein (VASP) and significantly increased endothelial permeability. Glutamate-induced decreases in brain endothelial barrier function and phosphorylated VASP were significantly attenuated by pretreatment of human brain endothelia with selective mGluR antagonists. These observations were extended to an in vivo hypoxic mouse model in which pretreatment with mGluR antagonists significantly decreased fluorescein isothiocyanate-dextran flux across the blood-brain barrier. We conclude that activated human PMNs release glutamate and that endothelial expression of group I or III mGluRs function to decrease human brain endothelial VASP phosphorylation and barrier function. These results identify a novel pathway by which PMN-derived glutamate may regulate human endothelial barrier function.
Collard
Charles D
CD
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Park
Kellie A
KA
Montalto
Michael C
MC
Alapati
Sailaja
S
Buras
Jon A
JA
Stahl
Gregory L
GL
Colgan
Sean P
SP
D001483:Base Sequence; D001812:Blood-Brain Barrier; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D018698:Glutamic Acid / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D009504:Neutrophils / Q000378:metabolism*; D017470:Receptors, Glutamate / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D017470:Receptors, Glutamate; D018698:Glutamic Acid